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REGENERATION  OF  MEDULLATED  NERVES  IN 
THE  ABSENCE  OF  EMBRYONIC  NERVE 
FIBERS,  FOLLOWING  EXPERIMENTAL  NON- 
TRAUMATIC DEGENERATION 


A  DISSERTATION 

SUBMITTED   TO   THE   FACULTY 

OF   THE   OGDEN   GRADUATE   SCHOOL   OF   SCIENCE 

IN   CANDIDACY   FOR   THE   DEGREE   OF 

DOCTOR   OF   PHILOSOPHY 

DEPARTMENT  OF  ANATOMY 


BY 

ELBERT  HOWARD  CLARK 


Private  Edition,  Distributed  By 

THE  UNIVERSITY  OF  CHICAGO  LIBRARIES 

CHICAGO,  ILLINOIS 


Reprinted  from 

The  Journal  of  Comparative  Neurology,  Vol.  XXIV,  No.  i 

February,  1914 


TEbe  tUnivetsits  ot  Cbicago 


REGENERATION  OF  MEDULLATED  NERVES  IN 
THE  ABSENCE  OF  EMBRYONIC  NERVE 
FIBERS,  FOLLOWING  EXPERIMENTAL  NON- 
TRAUMATIC DEGENERATION 


A  DISSERTATION 

SUBMITTED  TO   THE   FACULTY 

OF   THE   OGDEN   GRADUATE   SCHOOL   OF   SCIENCE 

IN   CANDIDACY   FOR   THE   DEGREE   OF 

DOCTOR   OF    PHILOSOPHY 

DEPARTMENT  OF   ANATOMY 


BY 

ELBERT  HOWARD  CLARK 


Private  Edition,  Distributed  By 

THE  UNIVERSITY  OF  CHICAGO  LIBRARIES 

CHICAGO,  ILLINOIS 


Reprinted  from 

The  Journal  of  Comparative  Neurology,  Vol.  XXIV,  No.  i 

February,  19 14 


Q?33| 
C5^ 


Reprinted  from  The  Journal  of  Comparative  Neurology,  Vol.  24,  No,  1 
February,  1914 


REGENERATION  OF  MEDULLATED  NERVES  IN  THE 
ABSENCE  OF  EMBRYONIC  NERVE  FIBERS, 
FOLLOWING  EXPERIMENTAL  NON- 
TRAUMATIC DEGENERATION 

ELBERT  CLARK 

From  The  Anatomical  Laboratory,  University  of  the  Philippines 

THIRTY-TWO   FIGURES 

INTRODUCTION 

The  present  study  is  based  upon  experiments  in  which  degen- 
eration and  regeneration  of  medullated  nerve  fibers  were  brought 
about  under  new  experimental  conditions.  The  results  obtained 
relate,  for  the  most  part,  to  phases  of  the  subject  upon  which 
the  evidence  has  heretofore  been  incomplete.  In  this  investi- 
gation, an  experimental  obstacle  which  has  been  responsible  for 
the  strikingly  contrary  observations  between  the  supporters  of 
auto-regeneration  on  the  one  hand  and  the  advocates  of  an  out- 
growth of  the  axis  cylinder  on  the  other,  has  been  entirely  avoided. 
I  refer  to  an  ingrowth  of  foreign  nerve  fibers  through  the  scar 
tissue  into  a  regenerating  medullated  nerve.  This  obstacle  was 
avoided  by  inducing  degeneration  in  the  peripheral  medullated 
nerves  of  the  domestic  fowl  by  a  prolonged  exclusive  feeding  of 
polished  rice,  and  subsequent  regeneration  by  a  return  to  an 
adequate  nutritive  diet. 

In  1897  Eijkman  first  described  'polyneuritis'  in  fowls  which 
had  been  kept  for  three  or  four  weeks  on  an  exclusive  diet  of 
polished  rice.  This  has  since  been  confirmed  by  numerous  other 
investigators  and  Frazer  and  Stanton  ('11)  have  noted  and  illus- 
trated 'Wallerian  degeneration'  in  the  nervus  ischiadicus  of  the 
domestic  fowl  which  developed  paralysis  on  a  polished  rice^  diet. 

^  White  rice,  polished  rice  or  decorticated  rice  is  the  clear  white  table  rice 
of  commerce.     It  is  rice,  which,  after  having  the  husk  taken  off,  is  further  sub- 

61 


62  ELBERT  CLARK 

In  another  place-  I  have  described  more  in  detail  the  changes 
occurring  in  the  nervous  system  of  such  fowls  Here  it  was 
also  pointed  out,  in  agreement  with  Frazer  and  Stanton  and 
others,  that  "The  neuritis  produced  in  fowls  by  a  prolonged  diet 
of  polished  rice  is,  so  far  as  the  best  evidence  indicates,  a  neuritis 
due  to  a  deficiency  of  some  food  constituent  or  constituents 
necessary  for  the  maintenance  of  the  metabolic  and  functional 
activity  of  the  nervous  system. "^ 

In  the  paralysis  of  fowls  brought  about  by  an  exclusive  diet 
of  polished  rice  the  medullated  fibers  of  the  sciatic  undergo  a 
rapid  degeneration.  This  degeneration,  however,  is  much  slower 
than  that  produced  as  a  result  of  transection  of  the  nerve.  More- 
over, for  the  rice-fed  fowls,  the  following  conditions  obtain:     In 

jected  to  a  process  of  'milling'  or  polishing.  "In  this  process  the  fruit  wall  or 
pericarp,  the  layers  subjacent  to  it  (the  subpericarpal  layers)  as  well  as  the 
embryo  are  removed,"  Frazer  and  Stanton  ('11).  These  authors  give  the  fol- 
lowing as  the  average  composition  of  polished  and  unpolished  rice: 


CARBO- 
BTDBA.TE 


per  cent 

Polished  rice j      7 .7 

Unpolished  rice I      9.0 


per  cent         per  cent 


0.25 
1.65 


77.23 
75.52 


ASH  j    MOISTURE 


per  cent     '     per  cent 

0.25  14.3 

1.08  12.75 


Unpolished  rice  or  red  rice  is  rice  which  has  not  been  subjected  to  the  polish- 
ing process,  and  which  as  a  consequence  still  has  the  pericarp,  subpericarpal 
layers  and  the  embryo.  Fowls  fed  exclusively  upon  unpolished  rice  for  long  peri- 
ods never  develop  neuritis  as  when  fed  exclusively  upon  polished  rice.  Further, 
neuritis  in  fowls  as  the  result  of  an  exclusive  rice  diet  can  most  frequently  be 
cured  by  placing  the  fowl  on  an  exclusive  diet  of  unpolished  or  red  rice.  There 
are  several  qualities  of  white  rice,  which,  aside  from  the  quality  of  the  grain, 
are  denoted  by  the  amount  of  polishing  to  which  the  rice  has  been  subjected. 
As  might  be  expected  the  most  highly  polished  grade  is  the  most  effective  in 
producing  paralysis  in  the  fowl. 

"  Edward  B.  Vedder  and  Elbert  Clark.  A  study  of  polyneuritis  gallinarum. 
Philippine  Journal  of  Science,  vol.  7,  no.  5,  Sec.  B,  p.  423. 

^  Richard  P.  Strong  and  B.  C.  Crowell  have  produced  experimentally  in  man 
a  similar  neuritis  by  the  prolonged  feeding  of  a  diet  of  which  polished  rice  formed 
by  far  the  main  constituent.  In  one  case  which  resulted  fatally,  the  peripheral 
nerves  showed  marked  degeneration  (The  etiology  of  beri-beri.  Phil.  Jour.  Sci- 
ence, B,  vol.  7,  p.  271).  John  M.  Little  has  also  described  beri-beri  in  man 
resulting  from  an  almost  exclusive  diet  of  white  bread  (Beri-beri  caused  by  fine 
white  flour.     Jour.  Amer.  Med.  Assoc,  vol.  58,  p.  2029). 


DEGENERATION  AND  REGENERATION  OF  NERVES      63 

degeneration  the  fibers  are  intact  and  all  traumatic  and  inflam- 
matory effect  produced  by  cutting  the  tissues  and  the  nerve  or 
of  tying  the  latter  are  obviated;  the  process  of  degeneration  can 
be  stopped  at  almost  any  stage  or  greatly  prolonged,  and  several 
stages  of  degeneration  are  to  be  observed  in  different  fibers  of 
the  same  nerve.  In  regeneration,  the  possibility  of  an  ingrowth 
of  fibers  from  other  nerves  into  the  regenerating  nerve  under 
observation  is  obviated  and  recovery  of  the  animal  can  be  accom- 
plished after  any  stage  of  degeneration  of  the  peripheral  nerves. 
And  lastly,  the  slowness  of  the  cycle  of  degeneration  and  recov- 
ery, makes  it  possible  to  draw  a  sharper  distinction  between  the 
process  of  degeneration  and  regeneration  in  medullated  nerve 
fibers.  At  the  present  time  these  experimental  conditions  are 
especially  desirable. 

PARALYSIS  IN  FOWLS  RESULTING  FROM  AN  EXCLUSIVE  DIET  OF 

POLISHED  RICE 

A  typical  case  of  a  fowl  which  has  become  unable  to  walk 
after  a  diet  of  polished  rice  is  found  in  that  of  No.  54  whose 
history  is  as  follows:  No.  54,  brown  hen,  fed  polished  rice  since 
February  7,  1912;  25  days  later,  on  March  3,  the  first  definite 
signs  of  unsteadiness  in  the  legs  were  noted.  March  4,  the  bird 
was  found  balanced  on  its  'haunches,'  was  scarcely  able  to  rise 
and  could  not  take  more  than  two  or  three  haphazard  steps 
without  tumbling  over  in  a  heap.s  The  diet  was  changed  to  a 
'regenerative'  diet  consisting  of  whole  grain,  meat  scraps,  bread, 
grass,  etc.  March  14,  could  stand  up  very  unsteadily  for  a  few 
seconds  but  was  scarcely  able  to  take  a  step.  March  20,  same 
— never  stood  up  nor  attempted  to  walk  unless  forced  and  as- 
sisted in  this.  April  3,  good  general  appearance  but  was  scarcely 
able  to  walk.  April  10,  improved,  but  walked  with  much  diffi- 
culty. May  3,  apparently  entirely  recovered  within  the  last  few 
days. 

It  should  be  noted  that  many  fowls  on  the  polished  rice  diet 
lose  complete  control  of  the  lower  part  of  the  legs.  Others  show 
wing  drop,   droopiness  of  the  head  and  inability  to  swallow. 


64  ELBERT   CLARK 

Still  others  show  complete  collapse."*  The  greatest  variety  of 
sjrmptoms  are  manifested  by  various  birds,  but  loss  of  control 
of  the  legs  is  the  most  frequent.  Fowls  showing  the  latter  syrap- 
tom,  with  otherwise  fair  to  good  general  condition,  were  the  ones 
selected  for  this  study;  nerves  of  these  show  a  more  pronounced 
degeneration,  and  recovery  in  this  class  of  fowls  is  more  easily 
accomplished.  Twenty  to  thirty  days  on  the  white  rice  diet  is 
the  usual  length  of  time  before  symptoms  of  neuritis  are  mani- 
fested. Some  birds  resist  for  35  or  40  days,  and  two  fowls^  that 
were  receiving  a  small  quantity  of  calcium  lactate  with  the 
rice  did  not  'come  down'  till  the  fifty-first  and  sixtieth  day 
respectively.  Nitrogenous  and  fatty  foodstuffs  in  very  small 
amounts  added  to  the  rice  also  greatly  defer  the  development 
of  the  neuritis.  For  more  complete  data  on  this  interesting  affec- 
tion and  for  feeding  experiments,  reference  should  be  made  to 
the  recent  article  by  Vedder  and  Clark  ('12). 

DEGENERATION 

A  few  remarks  should  be  made  at  this  point  concerning  the 
nature  and  extent  of  the  degeneration  in  the  medullated  fibers 
of  the  sciatic  nerve  in  fowls  of  the  class  under  consideration. 
In  the  nerves  of  60  chickens,  which  had  been  fed  20  days  or 
more  on  an  exclusive  diet  of  polished  rice,  degeneration  in  the 
fibers  of  the  sciatic  nerve  was  observed  by  the  aid  of  the  Marchi 
method  in  every  case  regardless  of  what  symptoms  were  mani- 
fested by  the  fowl  before  death.  Many  of  these  were  confirmed 
by  the  Weigert  method  for  staining  the  myelin  sheath.  Several 
fowls  fed  as  long  as  35  to  40  days  showed  no  signs  of  weakness 
in  the  legs  but  well  marked  nerve  degeneration.  The  nerves 
from  each  of  twelve  fowls  fed  from  7  to  22  days  consecutively 

*  Several  workers  liave  observed  that  fowls  occasionallj'  do  not  lose  weight 
on  the  polished  rice  diet.  Frazer  and  Stanton  ('11)  who  have  kept  very  comi)lete 
records  report  several  fowls  which  kept  their  weight  up  for  as  long  as  3.5  days. 
Other  fowls  even  showed  a  gain  in  weight. 

*  Courtesy  of  Dr.  R.  B.  Gibson  of  the  Department  of  Phj'siology;  from  experi- 
ments being  conducted  by  him  to  study  the  influence  of  an  addition  of  various 
salt  mixtures  to  the  white  rice  on  the  production  of  this  affection,  to  be  reported 
shortly. 


DEGENERATION  AND  REGENERATION  OF  NERVES      65 

with  no  leg  weakness  showed,  by  the  same  methods,  myeUn 
degeneration  in  their  fibers.  It  was  a  constant  observation  that 
different  fibers  of  a  given  nerve  present  the  greatest  variation  in 
the  degree  of  their  degeneration.  In  two  fowls  killed  after 
feeding  7  days  on  white  rice,  small  areas  of  blackening  after 
treatment  by  the  Marchi  method  were  observed  in  approximately 
one- third  of  the  fibers  of  the  sciatic.  These  areas  very  seldom 
involved  the  entire  diameter  of  the  individual  fiber  at  any  one 
point  and  the  great  majority  ranged  from  1  to  8  microns  in 
diameter  (fig.  32). 

In  the  sciatic  nerve  of  those  fowls  fed  for  a  longer  time  and 
which  developed  a  typical  paralysis  in  the  legs,  every  fiber  showed 
larger  areas  of  blackening.  Advanced  degeneration  was  found  in 
from  10  per  cent  to  20  per  cent  of  the  fibers.  The  change  shown 
by  these  latter  fibers  presents  an  identical  picture  of  degenera- 
tion with  that  in  medullated  fibers  of  a  mammalian  nerve  10  to 
14  days  after  section,  but  for  the  nuclei  of  the  neurilemma  sheath. 
The  nuclei  of  the  neurilemma  sheath  have  undergone  little  or 
no  multiplication.  This  will  be  again  referred  to  and  more  fully 
discussed  in  the  consideration  of  the  "embryonic  nerve  fiber." 
By  both  the  Marchi  method  and  the  Weigert  method  for  the 
medullary  sheath,  the  change  of  the  medullary  sljeath  substance 
into  fatty  globules  and  droplets  appears  complete  in  some  fibers. 
As  I  have  pointed  out  in  a  previous  paper  ('12), 

Fibers  showing  advanced  degeneration  are  marked  by  accumulation 
of  degenerated  myelin  in  large  globules  and  droplets,  a  swelling  and 
bulging  of  the  nerve  sheath  at  these  points  and  a  disintegration  of  the 
axis  cylinder.  The  largest  globules  usually  appear  vesicular  and  in 
their  center,  segments  or  fragments  of  the  axis  cylinder  are  frequently 
to  be  seen.  In  these  larger,  and  in  some  of  the  smaller  globules  also, 
the  stainable  material  is  found  at  the  periphery  and  appears  lami- 
nated. This  laminateci  appearance  is  very  characteristic  in  Weigert 
preparations  and  is  the  rule  in  the  larger  globules.  Usually  3  distinct 
layers  are  clearly  visible,  of  which  the  outer  is  the  thickest.  Other 
incomplete  layers  and  fragments  are  seen  centrally. 

But  for  an  increase  in  the  number  of  nuclei  of  the  neurilemma 
sheath,  this  description  applies  with  equal  exactness  to  fibers 
of  similar  preparations  of  the  peripheral  segment  of  the  sciatic 
nerve  of   a  fowl  7  days  after  section.     Figures  2,  3,  4  and  5 

THE  JOURNAL  OF  COMPARATIVE  NEUROLOGY,  VOL.  24,  NO.  1 


66  ELBERT   CLARK 

illustrate  the  marked  degeneration  described,  and  figure  6  shows 
segments  of  axis  cylinders  enclosed  within  large  globules. 

Howell  and  Huber  ('92),  Bethe  ('07),  Mott,  Halliburton  and 
Edmonds  ('04),  Cajal  ('05),  Ranson  ('12),  and  others  have  observed 
that  after  section  not  all  meduUated  fibers  degenerate  with  equal 
rapidity.  As  noted,  this  is  particularly  true  of  degeneration  in  the 
fibers  of  the  sciatic  in  fowls  on  a  white  rice  diet.  In  the  nerves 
of  these  fowls,  a  fiber  showing  the  first  indication  of  degener- 
ation may  be  found  side  by  side  with  one  in  which  the  neuraxis 
and  medullary  sheath  are  completely  broken  up  (fig.  7). 

These  few  remarks,  with  the  accompanying  figures  2  to  7, 
make  it  clear  that  paralysis  in  the  rice-fed  fowls  presents  an 
experimental  condition  resulting  in  degeneration  in  which  almost 
every  stage  of  the  process  is  to  be  observed  in  the  same  nerve 
at  one  and  the  same  time,  in  which  the  continuity  of  the  fibers 
is  not  disturbed,  where  reaction  to  trauma  is  obviated  and  where 
degeneration,  though  proceeding  rather  rapidly,  is  much  slower 
than  after  section  of  the  nerve. 

THE  EMBRYONIC  NERVE  FIBER 

Rapid  multiplication  of  the  nuclei  of  the  sheath  of  Schwann, 
coincident  witl\  the  change  in  the  medullary  sheath  and  axis 
cylinder,  has  been  a  constant  finding  with  all  those  observers 
who  have  studied  degeneration  of  nerves  after  section.  It  was 
a  little  surprising  to  find  it  not  to  obtain  in  the  present  case 
which  in  every  other  respect  resembles  Wallerian  degeneration 
and  in  which  the  process  of  degeneration  is  also  rapid.  A  care- 
ful search  of  both  teased  and  sectioned  preparations  of  nerves, 
taken  at  time  of  paralysis  and  in  which  the  nuclei  were  well 
stained,  failed  to  reveal  any  marked  or  definite  increase  in  the 
number-  of  nuclei  of  the  neurilemma  sheath  or  any  structure 
which  could  be  definitely  identified  as  an  embryonic  nerve  fiber 
in  the  degenerated  fibers  of  a  single  fowl. 

An  explanation  of  this  marked  variation  was  of  course  required, 
and  inasmuch  as  fowls  frequently  recovered  after  the  most  marked 
paralysis  of  the  legs,  it  became  necessary  to  determine  if  regen- 
eiation  occurs  in  that  10  to  20  per  cent  of  fibers  which  show 


DEGENERATION  AND  REGENERATION  OF  NERVES       67 

such  marked  degeneration  after  a  prolonged  diet  of  white  rice. 
This  being  the  case,  it  would  then  be  desirable  to  know  whether 
regeneration  could  take  place  in  these  fibers  without  going  through 
that  stage  termed  'embryonic  nerve  fiber,'  'Bandfiber'  or  'pro- 
toplasmic band.'  For  a  more  complete  understanding  of  the 
significance  of  the  multiplication  of  the  nuclei  of  the  neurilemma 
sheath  and  the  embryonic  nerve  fiber,  I  sought  to  determine 
if  degeneration  in  medullated  nerve  fibers  without  multiphcation 
of  the  nuclei  of  the  neurilemma  sheath  could  be  brought  about 
by  any  other  experimental  means;  and  if  so,  could  regeneration 
be  accomplished  in  these  without  the  embryonic  nerve  fiber  stage? 
Further,  could  it  be  shown  that  the  increase  in  the  number  of 
nuclei  usually  observed  is  due  to  trauma  or  inflammatory  influ- 
ences or  to  an  infiltration  of  phagocytes?  And  lastly,  does  the 
embryonic  nerve  fiber  represent  a  stage  of  regeneration  or  degen- 
eration? An  answer  to  these  questions  would  manifestly  throw 
additional  Ught  upon  the  significance  of  the  increase  in  number 
of  nuclei  of  the  sheath  of  Schwann  and  of  the  embryonic  nerve 
fiber.  This  proved  to  be  a  most  attractive  phase  of  the  investi- 
gation. 

Atrophic  degeneration  without  multiphcation  of  the  nuclei  of 
the  sheath  of  Schwann  has  been  frequently  described  in  certain 
chronic  pathologic  conditions  lasting  many  weeks  or  months. 
In  the  present  case,  however,  where  globulation  and  breaking 
up  of  the  axis  cylinder  have  been  observed  as  early  as  the  nine- 
teenth day  of  the  white  rice  diet  and  where  the  first  evidences 
of  change  noted  was  on  the  seventh  day,  the  degeneration  (tak- 
ing place  in  12  days)  can  not  be  said  to  have  anything  in  common 
with  atrophy. 

It  soon  became  evident  on  histologic  examination  that  regen- 
eration does  occur  in  those  fibers  in  which  the  neuraxis  and 
medullary  sheath  have  broken  up.  Having  failed  to  find  in  the 
degenerated  fibers  a  single  embryonic  nerve  fiber  or  smy  marked 
or  definite  increase  in  the  number  of  nuclei  of  the  neurilemma 
sheath,  I  next  examined  nerves  from  fowls  at  various  periods 
during  regeneration.  With  this  end  in  view,  fowls  were  killed 
after  having  been  kept  on  a  regeneration  diet  for  4,  11,  13,  14, 


68  ELBERT   CLARK 

16,  19,  21  and  30  days  respectively.  The  fowl  which  had  been 
kept  for  30  days  on  the  nutritive  diet  showed  marked  improve- 
ment. In  addition  to  nerves  from  this  series,  segments  of  the 
sciatic,  cut  out  from  fowls  48,  56,  59  and  60  days  in  regeneration 
respectively,  were  examined  for  embryonic  nerve  fibers  and  multi- 
pUcation  of  the  nuclei  of  the  neurilemma  sheath.  These  four 
fowls  had  just  become  able  to  walk  again.  In  neither  teased 
nor  sectioned  preparations  of  the  nerves  of  all  these  fowls  were 
embryonic  nerve  fibers  to  be  found.  Nor  was  there  observed 
a  more  definite  indication  of  multiplication  of  the  nuclei  of  the 
neurilemma  sheath  than  was  seen  in  the  nerves  of  those  fowls 
killed  at  the  time  paralysis  developed.  This  lack  of  nuclear 
multipHcation  also  obtained  in  nerves  of  fowls  108,  125,  171  and 
275  days  in  regeneration. 

Attempts  to  produce  by  other  means  a  degeneration  in  the 
medullated  fibers  without  a  multiplication  of  the  nuclei  of  the 
sheath  of  Schwann  were  not  successful.  Freezing  a  small  por- 
tion of  the  sciatic  with  carbon  dioxide  snow,  treating  a  portion 
with  chloroform  vapor,  and  injecting  a  drop  or  two  of  chloro- 
form into  the  nerve,  when  successful  in  producing  degeneration, 
resulted  also  in  the  typical  and  rapid  increase  in  the  nuclei  of 
the  neurilemma  sheath  as  has  been  constantly  described  after 
section  or  ligature  of  the  nerve.  Less  violent  means  were  then 
adopted  in  that  rubber  bands,  tight  enough  to  cut  off  the  circu- 
lation but  not  tight  enough  to  do  mechanical  injury  to  the  nerve 
were  placed  around  the  thigh  of  fowls.  Loss  of  control  of  the 
leg  resulted  after  3  or  4  days,  both  when  the  bandage  was  allowed 
to  remain  for  24  hours  at  a  time  and  when  it  was  on  and  off 
every  few  hours  over  a  course  of  2  or  3  days.  Although  by  this 
method  the  result  sought  for  was  not  accomplished,  yet  another 
observation  of  equal  importance  was  made.  Fowl  No.  78,  on 
which  the  bandage  was  allowed  to  remain  for  24  hours,  experi- 
enced considerable  oedema  of  the  bandaged  leg;  this  condition 
progressed,  without  infection,  to  dry  gangrene  of  nearly  all  of 
that  portion  peripheral  to  the  bandage  by  the  twenty-eighth 
day,  or  approximately  24  days  after  loss  of  control  of  the  leg. 
This,  then,  represents  a  nerve  in  which  only  retrogressive  changes 


DEGENERATION  AND  REGENERATION  OF  NERVES       69 

could  have  taken  place  as  gangrene  early  manifested  itself-  -by 
the  twelfth  day  of  the  loss  of  control.  Fibers  from  that  portion 
of  the  leg  affected  by  gangrene  presented  an  appearance  from 
which  they  were,  readily  recognized  as  embryonic  nerve  fibers 
(fig.  8).  For  the  most  part  these  were  very  slender  with  long 
nuclei,  an  appearance  which  is  readily  accounted  for  by  the  par- 
tial desiccation.  Many  still  contained  droplets  of  degenerated 
myelin.  It  is  difficult  to  see  how  the  least  regenerative  reaction 
could  have  taken  place  in  this  nerve. 

As  stated  above,  I  have  not  observed  a  single  instance  in  which 
there  was  a  marked  increase  in  the  number  of  nuclei  of  the  sheath 
of  Schwafm  in  those  degenerated  fibers  of  fowls  which  showed 
paralysis  of  the  legs  after  20  to  30  days  on  polished  rice.  In 
fibers  showing  the  most  advanced  degeneration,  that  is,  marked 
globulation  of  myelin  and  breaking  up  of  axis  cylinder,  measure- 
ments were  made  between  neighboring  nuclei  of  the  sheath  of 
Schwann.  Among  these  measurements,  there  were  observed  such 
distances  as  these  between  successive  nuclei:  368,  386,  477  and 
379  microns  respectively.  The  distance  between  2  nodes  of 
Ranvier  is  variously  estimated  as  from  80  At  to  900 fj,  according 
to  the  diameter  of  the  fiber,  and  ''in  the  higher  vertebrates  a 
single  nucleus  is  found  midway  between  each  two  nodes" — Huber. 
Mitosis  has  not  been  observed. 

Some  few  fibers,  however,  were  seen  in  which  the  nuclei  of 
the  sheath  of  Schwann  occurred  at  intervals  frequent  enough  to 
be  suggestive  of  a  slight  increase  in  their  number.  This  was 
suggestive  enough  to  make  it  desirable  to  examine,  for  an  in- 
crease of  the  number  of  nuclei  of  the  sheath  of  Schwann,  nerves 
of  fowls  with  which  the  onset  of  paralysis  had  been  deferred  by 
the  addition  of  very  small  amounts  of  other  foodstuffs  to  the 
rice.  At  this  period  I  was  fortunate  in  securing  from  Dr.  R. 
B.  Gibson  of  the  Department  of  Physiology,  a  fowl.  No.  17,  G, 
which  suddenly  developed  paralysis  of  the  legs  after  60  days  on 
a  diet  of  white  rice  and  calcium  lactate.  This  fowl  developed 
a  typical  case  of  leg  paralysis  two  days  before  death.  Marchi 
preparations  of  the  sciatic  showed  very  extensive  and  advanced 
myelin  degeneration. 


70  ELBERT   CLARK 

Teased  preparations  from  the  sciatic  of  this  fowl  stained  to 
bring  out  the  nuclei  revealed  a  few  fibers  of  the  embryonic  nerve 
fiber  type.  These  were  very  slender  fibers  with  nuclei  at  fre- 
quent intervals.  Protoplasm  was  scant  even  around  the  nuclei, 
though  the  structures  stained  well  (fig.  9).  Many  resembled 
very  closely  non-medullated  nerves  in  degeneration  described  by 
Ranson  ('12).  Others  were  found,  however,  which  contained  a 
larger  amount  of  protoplasm  and  in  which  small  droplets  of 
degenerated  myelin  could  occasionally  be  found  (fig.  10).  This 
observation  clearly  shows  these  to  have  been  derived  from  medul- 
lated  nerve  fibers.  No  axis  cylinder  could  be  demonstrated  by 
special  stains.  Another  fowl,  No.  9,  G,  on  a  similar  diet  and 
with  a  somewhat  similar  history  developed  paralysis  of  the  legs 
after  51  days.  Teased  preparations  from  the  sciatic  of  this  fowl, 
when  stained  to  bring  out  the  nuclei,  also  showed  embryonic 
nerve  fibers.  These  were  a  little  more  numerous  than  in  No. 
17,  G,  were  larger  and  contained  niore  protoplasm.  Protoplasmic 
granules  were  seen  in  the  immediate  neighborhood  of  the  nucleus 
and  droplets  of  degenerated  myelin  were  of  rather  frequent  oc- 
currence. Many  of  the  fibers  bore  an  exact  resemblance  to  the 
protoplasmic  bands  to  be  seen  in  meduUated  nerves  after  section. 
Figure  11,  which  is  from  a  teased  preparation  of  the  sciatic  nerve 
of  No.  9,  G,  illustrates  this  resemblance  and  shows  beyond  ques- 
tion their  identity  with  the  embryonic  nerve  fibers  of  a  sec- 
tioned nerve. 

Fowl  No.  57,  which  came  down  with  severe  paralysis  and 
prostration  after  23  days  on  a  diet  of  polished  rice  and  which 
made  a  most  difficult  recovery,  was  killed  1  year  and  14  days 
after  being  placed  on  a  regeneration  diet  and  approximately  10 
months  after  all  symptoms  of  paralysis  had  disappeared.  Teased 
preparations,  stained  as  above,  showed  among  several  thousand 
fibers  one  very  frail  fiber  with  nuclei  at  frequent  intervals.  The 
fiber  appeared  scarcely  more  than  a  strand  of  connective  tissue 
with  well  staining  spindle-shaped  nuclei  along  its  course.  This 
was  probably  an  embryonic  nerve  fiber.  Previous  observations 
had  convinced  me  that  regeneration  of  the  axis  cylinder  fails 
to  take  place  in  a  very  small  percentage  of  those  fibers  which 


DEGENERATION  AND  REGENERATION  OF  NERVES       71 

have  shown  degenerative  changes.  This  slender  fiber  with  fre- 
quent nuclei,  then,  might  easily  represent  a  later  stage  or  atrophy 
of  a  fiber  which  failed  of  regeneration. 

To  summarize  briefly:  Neither  embryonic  nerve  fibers  nor  a 
marked  multiplication  of  the  nuclei  of  the  neurilemma  sheath 
have  been  found  in  degenerating  fibers  of  fowls  showing  paralysis 
after  20  to  30  days  on  polished  rice:  they  have  not  been  observed 
(with  one  possible  exception)  in  regenerating  fibers  of  fowls  re- 
covering from  this  paralysis:  embryonic  nerve  fibers  containing 
droplets  of  degenerated  myelin  were  found  in  the  nerves  of  fowls 
fed  for  51  and  60  days  on  a  polished  rice  diet,  and  embryonic 
nerve  fibers  entirely  replaced  the  nervous  elements  in  the  sciatic 
of  a  fowl  whose  leg  was  undergoing  progressive  necrosis. 

This  chain  of  evidence  seems  complete  and  can  leave  little 
doubt  as  to  the  significance  of  the  so-called  embryonic  nerve 
fiber,  Band-faser  or  protoplasmic  band.  In  each  instance  except 
the  last  we  have  the  embryonic  nerve  fiber  occurring  in  a  nerve 
which  is  undergoing  progressive  retrogression.  In  fowl  No.  78 
the  degeneration  was  due  to  mechanical  causes.  In  No.  17,  G, 
and  No.  9,  G,  those  few  fibers  which  always  showed  marked 
degeneration  by  the  thirtieth  day,  regardless  of  whether  S3rmp- 
toms  are  manifested  or  not,  had  had  time  for  a  more  advanced 
degeneration  than  those  fibers  of  fowls  killed  at  an  earlier  date. 
In  either  case,  it  was  impossible  for  regeneration  to  be  taking  place. 

Thus  the  conclusion  that  the  embryonic  nerve  fiber  represents 
a  late  stage  of  degeneration  is  a  logical  one.  Degeneration  as 
used  above  is  meant  to  imply  a  retrogressive  change  in  the  myehn 
sheath  and  axis  cylinder.  It  is  to  be  noted  that  multiplication 
of  the  nuclei  of  the  neurilemma  sheath  and  the  resulting  embry- 
onic nerve  fiber  appear  only  after  degeneration  in  the  axis  cylin- 
der and  medullary  sheath  has  advanced  to  a  late  stage.  As  Van 
Gehuchten  points  out,  the  sequence  of  events  in  the  formation 
of  the  embryonic  nerve  fiber  with  cytoplasm  and  cytoplasmic 
granules  around  the  nuclei  must  be  considered  evidence  of  proto- 
plasmic activity  and  in  themselves  bear  a  close  resemblance  to 
regenerative  processes.  This,  however,  does  not  necessarily  imply 
an  attempt  on  the  part  of  the  structure  at  formation  of  a  new 


72  ELBERT   CLARK 

medullary  sheath  or  axis  cylinder.  Indeed,  Mott,  Halliburton 
and  Edmonds  ('04)  in  regeneration  in  nerves  after  section,  find 
that  the  new  medullary  sheath,  as  well  as  the  axis  cylinder, 
progresses  from  the  point  of  union  of  central  and  peripheral 
stumps.  I  will  suggest  that  the  multiplication  of  the  nuclei  of 
the  neurilemma  sheath  and  the  formation  of  the  embryonic  nerve 
fiber  in  the  rice-fed  fowls  is  comparable  to  the  proliferation  of 
connective  tissue  in  organs  undergoing  atrophj^  or  degeneration 
from  other  causes.  In  the  fowls  degeneration  of  the  nerve  fibers 
is  ;low  and  it  is  probable  that  the  stimulus  is  not  sufficient  to 
bring  about  a  multiplication  of  the  neurilemma  nuclei  until  very 
late.  On  the  other  hand,  the  trauma  occasioned  by  transsection 
of  the  nerve  introduces  a  violent  reaction  and  multiplication  of 
the  nuclei  of  the  neurilemma  sheath  rapidly  ensues. 

INFILTRATION   OF   PHAGOCYTES   AND   ABSORPTION   OF  THE 
DEGENERATED  MYELIN 

Stroebe  ('93),  Mott,  Halliburton  and  Edmonds  ('04),  Nageotte 
('11)  and  other  have  described  an  infiltration  of  phagocjiiic 
"wandering  cells  into  medullated  fibers  undergoing  myelin  degen- 
eration. Nageotte  ('11)  claims  it  as  a  constant  occurrence  and 
sees  in  their  presence  a  means  of  removal  of  the  degenerated 
myelin.    According  to  Nageotte  ('11),  these  foreign  elements 

constitute  the  agents  of  greatest  activity  in  the  resorption  of  the  de- 
generated myelin.  This  does  not  signify  that  the  syncytium  of  Schwann 
remains  inert;  it  can,  indeed,  resorb  the  myelin,  and  it  is  probable  that, 
in  the  fine  fibers,  it  accomplishes  the  work  of  phagocyotosis  of  the 
mylein  without  the  aid  of  the  foreign  elements.  In  the  large  fibers, 
at  the  end  of  the  third  day,  one  sees  in  the  perinuclear  protoplasmic 
mass  special  granulations  which  result  from  the  disintegration  of  the 
myelin;  but,  in  these  fibers  the  larger  part  of  the  myelin  becomes  the 
prey  of  the  leucocytes.  It  is  probable  that  the  leucocytes  emigrate 
once  their  work  is  accomplished. 

In  nerves  of  fowls  with  marked  leg  paralysis  after  about  30 
days  on  polished  rice,  I  have  sometimes  seen  fibers  in  degener- 
ation in  which  the  appearance  of  infiltration  by  wandering  cells 
is  very  striking.  These  cells  were  all  very  small  and,  after  Miiller 
fixation,  scarcely  anything  but  the  nucleus  was  to  be  seen  in 


DEGENERATION  AND  REGENERATION  OF  NERVES      73 

hematoxylin  and  carmine  preparations.  In  an  occasional  fiber 
they  were  quite  numerous,  though  uniformly  absent  from  those 
fibers  showing  the  larger  globules  of  degenerated  myelin  with 
enclosed  segments  of  the  axis  cylinder.  Figure  12  shows  the 
most  marked  instance  of  infiltration  observed  in  this  series.  Here 
it  is  clearly  evident  that  not  more  than  one  nucleus  belongs  to 
the  sheath  of  Schwann.  These  nuclei  have  not  been  observed 
in  fibers  from  fowls  which  developed  paralysis  after  51  days  or 
more  on  polished  rice.  Their  presence  in  some  fibers,  however, 
is  sufficient  to  show  that  trauma  is  not  alone  responsible  for  their 
presence. 

I  have  not  been  able  to  determine  the  significance  of  these 
wandering  cells  or  their  fate.  That  their  participation  in  the 
removal  of  the  degenerated  myehn  is  improbable  and  at  most 
can  be  only  extremely  shght,  is  clearly  manifested  by  the  per- 
sistence of  degenerated  myelin  seen  in  fibers  of  Marchi  prepa- 
rations from  fowls  which  had  been  kept  a  long  time  on  a  re- 
generation diet  and  for  several  months  after  all  symptoms  of 
paralysis  had  disappeared. 

In  nerves  from  these  fowls,  both  small  droplets  and  large  glob- 
ules of  degenerated  myelin  still  obtained,  and  indeed  in  some 
fibers  the  picture  resembled  very  closely  that  of  degeneration  in 
medullated  nerves  of  fowls  but  recently  afTected  by  paralysis. 
There  seemed  to  have  been  some  httle  absorption  of  degenerated 
myelin  in  the  nerves  of  those  birds  kept  for  4  months  in  regen- 
eration, yet  globulation  was  just  as  marked  and  the  globules 
v/ere  equally  large.  The  globules  of  degenerated  myelin  in  the 
fibers  of  fowls  dead  just  after  paralysis  appeared  dense,  while 
in  the  fibers  of  fowls  4  months  in  regeneration  the  globules  fre- 
quently appeared  honey-combed  or  somewhat  reticulated.  The 
droplets  of  degenerated  myelin  were  uniformally  smaller  in  the 
latter.  Fowl  No.  38  (leg  paralysis  after  22  days  on  polished 
rice,  recovery  apparently  complete  after  59  days  on  regeneration 
diet)  was  killed  on  the  108th  day  of  regeneration  or  49  days 
after  all  symptoms  of  neuritis  had  disappeared.  Every  fiber  of 
the  sciatic  nerve  of  this  fowl  contained  droplets  or  globules  of 
myelin  and  many  of  the  larger  globules  just  described.     Even 


74  ELBERT   CLARK 

the  swelling  of  the  fiber  and  out-bulging  of  the  sheath  at  the 
large  globules  obtained  and  the  larger  globules  were  still  vesicu- 
lar. Figure  13  is  a  photomicrograph  of  a  Marchi  preparation 
of  the  sciatic  nerve  from  this  fowl.  Droplets  of  degenerated  mye- 
lin are  clearly  visible  in  every  fiber  and  the  larger  globules  occur 
at  frequent  intervals.  Figure  14  is  a  similar  picture  of  the  sciatic 
of  No.  54,  171  days  in  regeneration.  The  droplets  and  globules 
of  degenerated  myelin  are  equally  definite  here. 

Degenerated  myelin  was  found  to  persist  in  the  sciatic  nerves 
of  a  fowl  10  months  after  all  signs  of  paralysis  had  disappeared 
and  1  3^ear  and  14  days  after  regeneration  diet  was  started. 
Figure  15  is  taken  from  a  Marchi  preparation  of  the  sciatic  nerve 
of  No.  57,  whose  recovery  history  was  as  is  just  indicated.  The 
largest  globules  have  completely  disappeared  or  more  probably 
have  considerably  decreased  in  size.  The  smaller  droplets  are 
also  decidedly  less  numerous  and  the  total  amount  of  degenerated 
myelin  is  comparatively  small. 

In  view  of  the  finding  that  the  sensory  nerves  usually  show  an 
apparent  recovery  before  the  motor,  it  was  thought  worth  while 
to  compare  the  sensory  and  motor  portions  of  the  sciatic  nerve 
of  No.  57  (1  year  and  14  days  in  regeneration).  Accordingly  the 
motor  and  sensory  portions  of  one  of  the  larger  roots  were  sepa- 
rated for  some  distance  peripheral  to  the  dorsal  root  ganglion, 
each  stained  by  the  Marchi  method  and  teased.  No  difference 
whatever  could  be  distinguished  between  the  two.  It  was  quite 
impossible  to  tell  one  from  the  other  (compare  fig.  15  and  fig.  16). 

It  would  thus  seem  that,  in  the  present  case  at  least,  phago- 
cytes are  in  no  way  concerned  in  the  removal  of  degenerated 
myelin.  Phagocytosis  is  clearly  excluded  inasmuch  as  the  dis- 
appearance of  myelin  is  so  extremely  slow  and  since  wandering 
cells  are  as  a  rule  not  found  in  degenerating  nerve  fibers  of  the 
fowls  under  consideration. 

Just  why  degenerated  myelin  should  persist  so  long  in  nerve 
fibers  of  these  fowls  and  disappear  so  quickly  from  those  nerves 
which  have  been  sectioned  or  ligated  or  even  from  No.  78  of 
my  series,  whose  leg  had  been  bandaged  for  24  hours,  is  not 
clear.     The  above  observations  would  seem  to  exclude  removal 


DEGENEEATION  AND  REGENERATION  OF  NERVES       75 

by  wandering  cells  which  are  common  to  both.  An  explanation 
might  be  sought  in  the  multiplication  of  the  nuclei  of  the  sheath 
of  Schwann  and  the  resulting  embryonic  nerve  fiber,  which  are 
such  marked  characteristics  of  degeneration  in  the  latter,  while 
being  uniformally  absent  from  the  nerves  of  fowls  showing  an 
early  paralysis  after  a  white  rice  diet.  This  is  rendered  still 
more  probable  when  it  is  remembered  that  in  those  fowls  in 
which  paralysis  was  deferred — No.  17,  G,  and  No.  9,  G — typical 
embryonic  nerve  fibers  were  present  and  within  these  fibers  de- 
generated myehn  occurred  in  only  very  snaall  amounts  or  was 
entirely  absent.  These  two  fowls  differ  from  those  showing  pa- 
ralysis at  an  earlier  date  in  that  in  the  former  degeneration  in 
a  certain  proportion  of  the  fibers  had  progressed  to  a  later  stage 
than  with  those  coming  down  at  an  early  period.  Were  it  possi- 
ble to  keep  the  fowl  alive  on  the  white  rice  diet  till  all  fibers  were 
given  opportunity  to  undergo  advanced  degeneration,  I  have  no 
doubt  but  that  after  regeneration  in  the  same  animal  the  med- 
ullated  nerve  fibers  would  be  quite  devoid  of  even  droplets  of 
degenerated  myelin. 

In  those  fowls  that  have  recovered  from  paralysis  after  20  to 
30  days  on  poHshed  rice,  the  whole  chain  of  evidence  convinces 
me  that  here  degeneration  is  interrupted  in  the  middle,  as  it 
were;  and  regeneration  is  accomplished  or  superimposed  without 
passing  through  the  later  stages  of  degeneration.  This  being 
the  case,  we  must  attribute  the  rapid  removal  of  degenerated 
myelin  in  sectioned  nerves  to  the  activity  of  the  new  nuclei  of 
the  sheath  of  Schwann  and  the  embryonic  nerve  fiber.  This 
assumption  is  further  borne  out  'by  the  well-known  observations 
that  in  medullated  fibers  of  the  central  nervous  system  in  degen- 
eration the  globules  of  degenerated  myelin  persist  for  a  very 
long  time.  Halliburton  ('07),  in  speaking  of  this  point,  says: 
''In  situations  like  the  central  nervous  system  where  the  neuri- 
lemma is  non-existent,  not  only  is  the  removal  of  degenerated 
myelin  a  very  slow  process,  but  as  is  well  known,  regeneration 
does  not  occur,"  Schroder  ('08)  points  out,  in  speaking  of  de- 
generation in  medullated  fibers  of  the  cord:  "Noch  nach  einem 
Jahr  sind  grobe  SchoUen  sowie  namenthch  feine  Tropfchen  zu 


76  ELBERT   CLARK 

finden."  This  is  a  constant  finding  in  fibers  of  the  cord  unless 
special  precautions  are  taken  to  produce  marked  inflammation 
of  the  affected  area,  such  as  by  infection.  Then  the  degenerated 
myelin  is  removed  somewhat  more  rapidly,  though  even  here  not 
so  rapidly  as  in  the  peripheral  nerves  after  section.  With  pro- 
nounced inflammation  other  factors  are  introduced  which  would 
readily  account  for  the  more  rapid  removal  of  the  degenerated 
myelin;  as  they  have  no  bearing  on  the  question  they  need  not 
be  considered  here.  Now,  medullated  fibers  of  the  cord  differ 
histologically  from  medullated  fibers  of  the  peripheral  nerves  in 
that  the  former  do  not  possess  a  neurilemma.  After  section 
Wallerian  degeneration  of  the  one  differs  from  that  of  the  other 
only  in  that  the  proliferation  of  the  nuclei  of  the  neurilemma 
sheath  and  the  resulting  embryonic  nerve  fiber  are  lacking  in 
the  fibers  of  the  cord.  Infiltrating  phagocytes  are  found  in 
degeneration  in  both  cases. 

It  is  clear  then  that  the  rapid  multiplication  of  the  nuclei  of 
the  neurilemma  sheath  introduces  a  factor  which  is  responsible 
for  the  rapid  removal  of  the  degenerated  myelin.  It  is  probable 
that  the  protoplasmic  activity,  represented  by  the  multiplying 
nuclei  and  the  accumulation  of  protoplasm  around  these,  is  di- 
rectly concerned  with  the  rapid  resorption  of  the  degenerated 
myelin. 

I  have  no  evidence  suggestive  of  a  further  activity  of  the 
embryonic  nerve  fiber  and  its  nuclei  and  this  phase  of  the  sub- 
ject was  not  taken  up.  A  definite  zone  rich  in  protoplasm  was, 
however,  observed  around  the  nuclei  on  the  embryonic  nerve 
fibers  of  the  sciatic  of  fowl  No.' 9,  G.  In  this  protoplasm,  dis- 
crete granules  were  to  be  observed  in  preparations  stained  with 
Mallory's  phosphomolybdic  acid  hematoxylin  for  axis  cylinders.. 
These  granules  have  been  noted  by  Reich  and  others.  According 
to  Stroebe  ('93),  Nageotte  ('11)  and  others  they  bespeak  an 
activity  of  the  nuclei  concerned  in  the  development  of  a  new 
medullary  sheath. 

This  view  in  one  form  or  another  has  been  frequently  advo- 
cated, both  as  an  hypothesis  and  as  an  interpretation  of  the  fact 
that,  as  pointed  out  by  Stroebe   ('93),   the  protoplasm  which 


DEGENERATION  AND  REGENERATION  OF  NERVES      77 

increases  in  amount  with  the  multiphcation  of  the  nuclei  and 
the  disappearance  of  the  degenerated  myelin  in  nerves  after  sec- 
tion, contains  numerous  small  granules  and  droplets  of  fatty 
material.  Thus  according  to  Mott,  Halliburton  and  Edmonds 
('04),  ''they  (i.e.,  the  nuclei  of  the  neurilemma  sheath)  multiply, 
and  later  appear  to  share  with  phagocytes  in  the  removal  of  the 
broken  up  myelin  droplets."  And  Schroder  ('08),  basing  his 
views  upon  the  microscopic  observations  of  Stroebe  and  Biingner 
and  Schiitte,  has  the  following  to  say  relative  to  the  removal  of 
the  myelin  clumps: 

In  this  purely  degenerative  process  (i.e.,  the  early  clumping  of  the 
myelin)  early  progressive  occurrences  interpose  themselves.  The  nu- 
clei of  the  sheath  of  Schwann  begin  to  proliferate  already  on  the  second 
day,  according  to  Stroebe  they  attain  their  maximum  increase  in  num- 
ber through  mitosis  at  about  the  eighth  day;  coincident  with  the  pro- 
liferation of  the  nuclei  protoplasm  develops,  it  shoves  itself  into  the 
breaches  and  spaces  between  the  myelin  clumps,  flows  around  the 
clumps,  then  gathers  itself  together  into  a  single  round  or  oval,  demar- 
cated, single-,  or  many-celled  structure,  within  whose  interior  the 
clumps  of  myelin  rapidly  diminish  themselves  to  fine  granules  (accord- 
ing to  Biingner  and  Stroebe).  In  this  way  arise  frequently  granular 
cells  with  round  cell  body  and  a  fine  latticed  protoplasm  in  whose 
meshes  the  granules  lie  enclosed.  Such  elements,  from  about  the  fourth 
week  on,  are  to  be  found  in  the  lymph  spaces  around  the  neighboring 
vessels.  Stroebe  and  Schiitte  mention,  that  the  genesis  of  thse  granu- 
lar cells  has  been  often  incorrectly  conceived;  with  predilection,  one 
has  declared  them  leucocytes  or  so-called  wandering  cells  and  has 
assumed  that  they  arise  from  the  vessels  opened  at  the  point  of  the 
primary  injury  to  the  nerve,  move  forward  along  the  nerve  sheath  and 
then  take  up  at  all  places  the  disintegrated  myehn. 

REGENERATION 

Marked  multiphcation  of  the  nuclei  of  the  sheath  of  Schwann 
at  an  early  stage  of  degeneration  and  the  resulting  embryonic 
nerve  fiber  have  been  constant  findings  with  all  those  observers 
who  since  Waller,  have  studied  degeneration  and  regeneration 
of  medullated  nerves  after  section.  Upon  this  point  there  is 
complete  agreement.  The  interpretation,  however,  of  the  sig- 
nificance of  the  increased  number  of  nuclei  and  more  particularly 
of  the  embryonic  nerve  fiber  has  given  rise,  as  is  well  known,  to 
the  most  heated  controversies,  often  involving  personalties.     As 


78  ELBERT   CLARK 

a  result  the  most  varied  experiments  have  been  conducted  and 
a  vast  amount  of  evidence  on  all  possible  phases  of  the  subject 
has  been  presented.  Unfortunately,  however,  the  question  re- 
mains unsettled.  Unprejudiced  authors  of  text-books  still  include 
both  theories. 

Two  distinct  and  opposite  views  relative  to  regeneration  after 
the  embryonic  nerve  fiber  stage  are  at  present  current.  Accord- 
ing to  one,  most  vigorously  and  ably  advocated  by  Bethe,  the 
embryonic  nerve  fiber  is  capable  of  producing  per  se  a  new  medul- 
lary  sheath  and  new  axis  cylinder,  which,  later  making  connec- 
tion with  the  central  stump,  results  in  a  regenerated  and  func- 
tioning medullated  nerve  fiber;  in  young  animals  at  least,  the 
regenerated  fiber  is  capable  of  conducting  impulses  regardless  of 
whether  or  not  connection  with  the  central  stump  is  established. 
The  supporters  of  the  contrary  theory  claim  that  a  new  axis 
cylinder  for  the  peripheral  stump  is  attained  only  by  a  down 
growth  of  axis  cylinders  from  the  central  stump.  While  there 
is  some  difference  of  opinion  as  to  minor  points,  this  is  the  main 
contention  of  those  who  advocate  the  'outgrowth'  theory.  The 
sequence  of  events  as  interpreted  by  the  adherents  of  this  theory 
is  briefly  set  forth  by  Halliburton  ('07)  as  follows: 

From  the  microscopic  study  of  the  distal  portions  of  divided  nerve 
trunks,  we  arrived  at  the  conclusion  that  the  activity  of  the  neuri- 
lemma! cells  has  some  relation  to  the  development  of  new  nerve  fibers. 
At  an  early  stage  in  degeneration  their  nuclei  multiply;  later  they  par- 
ticipate with  phagocytes  in  the  removal  of  the  broken  up  myelin  drop- 
lets; subsequently  they  elongate  and,  becoming  connected  end  to  end, 
lead  to  the  formation  of  what  some  have  termed  "embryonic"  nerve 

fibers We  arrived  finally  at  the  conclusion  similar  to 

that  which  Howell  and  Huber  reached  fifteen  years  ago,  that,  although 
the  peripheral  structures  are  active  in  preparing  the  scaffolding,  the 
axis  cylinder  which  is  the  essential  portion  of  the  nerve  fiber  has  an 
exclusively  central  origin. 

Stroebe  ('93),  Huber  ('95),  Cajal  ('05),  Marienesco  ('06),  Ran- 
son  ('12)  and  others  have  described  and  illustrated  microscopic 
preparations  of  medullated  nerves  in  regeneration  after  section 
which  seem  to  show  beyond  a  doubt  that  outgrowth  of  the  axis 
cylinder  from  the  central  stump  does  take  place.  On  the  other 
hand  Bethe  ('02),  who  bases  his  opinion  mostly  upon  physio- 


DEGENERATION  AND  REGENERATION  OF  NERVES       79 

logical  grounds,  has  met  the  objections  of  his  opponents  in  a 
most  creditable  manner  by  repeated  experiments  and  new  evi- 
dence which  appear  irrefutable.  Langley  and  Anderson  ('04), 
Lugaro  ('05)  and  Mott,  Halliburton  and  Edmonds  ('04)  have 
repeated  many  of  Bethe's  experiments  but  oppose  rather  than 
support  him  in  his  contention  for  auto-regeneration  in  the  periph- 
eral stump,  Langley  and  Anderson  ('04)  admit  that  the  periph- 
eral stump,  after  a  sufficient  length  of  time  has  elapsed,  may  be 
found  capable  of  conducting  impulses  even  when  union  with  the 
central  stump  is  successfully  prevented.  They  explain  this  ap- 
parent auto-regeneration  by  an  ingrowth  of  nerve  fibers  from 
the  surrounding  tissues.  In  every  case  where  the  peripheral 
stump  became  capable  of  conducting  impulses,  strong  evidence 
was  obtained  to  show  that  an  ingrowth  of  fibers  into  it  from  other 
neighboring  nerves  had  taken  place.  Mott,  Halliburton  and 
Edmonds  ('04)  have  confirmed  this  finding  and  have  found  that 
in  a  piece  of  medullated  nerve  transplanted  in  the  same  animal 
in  such  a  manner  that  an  ingrowth  of  fibers  from  other  nerves 
is  prevented,  regeneration  fails  to  take  place.  They  have  further 
shown  that  in  a  regenerating  nerve  the  medullary  sheath  ''appears 
earliest  at  situations  near  the  point  where  the  ends  of  a  nerve 
have  been  joined  together,  and  reaches  the  distal  portions  later." 
Bethe  ('07)  has  again  repeated  these  experiments,,  but  can  find 
no  reason  to  abandon  his  former  strong  conviction  that  auto- 
regeneration  takes  place  in  the  peripheral  portion  of  a  divided 
medullary  nerve.  The  experimental  work  of  Ballance  and  Purves 
Stewart  ('01)  lead  them  to  declare  for  auto-regeneration,  and 
Van  Gehuchten  ('04)  has  confirmed  Bethe's  results.  Wilson  ('09)  , 
after  repeating  some  of  Bethe's  work,  draws  no  specific  conclusion 
regarding  auto-regeneration  of  divided  medullary  nerves. 

In  short  the  main  contention  of  the  advocates  of  auto-regen- 
eration hangs  on  whether  there  may  be  no  ingrowth  of  foreign 
fibers  from  neighboring  nerves  into  the  peripheral  portion  of  a 
divided  nerve.  This  at  bottom  is  the  point  in  dispute  between 
a  class  of  able  workers  who  hold  to  auto-regeneration  and  a 
group  of  equally  acute  observers  who  advocate  the  outgrowth 
of  the  axis  cylinder  from  the  central  stump.     So  long  as  so  cap- 


80  ELBERT   CLARK 

able  investigators  are  unable  to  obtain  the  same  result  in  a 
given  experiment,  just  so  long  will  our  theories  on  regeneration 
of  divided  medullated  nerves  remain  at  variance.  One  cannot 
help  but  suspect,  however,  that  on  account  of  this  very  differ- 
ence in  results  and  the  very  heated  controversy  on  the  subject, 
regeneration  of  medullated  nerves  after  section  has  received  more 
attention  and  investigation  that  the  comparative  importance  of 
the  subject  would  warrant. 

It  was  with  no  desire  to  engage  in  such  a  discussion  that  the 
present  work  was  begun,  and  the  new  points  brought  out  by  it 
in  regard  to  degeneration  will  greatly  outweigh  the  observations 
on  regeneration.  On  the  other  hand,  however,  I  considered  the 
controversy  of  others  no  excuse  for  avoiding  the  subject  when 
a  promising  experiment  was  thrown  at  my  door. 

As  stated  above,  in  fowls  fed  for  a  long  time  on  polished  rice, 
there  results  an  acute  degeneration  in  the  medullated  nerve  fibers 
resulting  in  a  breaking  up  of  the  myelin  into  large  globules  and 
droplets  and  a  segmentation  of  the  axis  cylinder.  This  change 
takes  place  in  from  12  to  18  days  and  bears  the  closest  resem- 
blance to  degeneration  in  medullated  nerve  fibers  after  section. 

Clearly  then,  if  it  could  be  shown  that  the  process  of -degen- 
eration in  the  nerve  fibers  of  the  rice-fed  fowls  was  identical  with 
degeneration  after  section  and  if  regeneration  takes  place  in  the 
former,  then  regeneration  of  medullated  fibers  was  accomplished 
without  the  possibility  of  an  ingrowth  of  fibers  from  other  nerves 
and  the  main  ground  for  a  difference  of  opinion  on  auto-regen- 
eration was  obviated.  If,  on  the  other  hand,  degeneration  in 
the  two  is  not  identical  nor  comparable,  it  would  still  be  of 
interest  to  know  if  regeneration  in  the  rice-fed  fowls  is  or  is  not 
concerned  with  the  so-called  'embryonic  nerve  fiber,'  'Band- 
faser'  or  'protoplasmic  band;'  if  auto-regeneration  obtains;  or  if 
the  new  axis  cylinder  results  from  an  outgrowth  of  the  central 
connection.  Whatever  the  result  arrived  at,  the  facts  collected 
from  this  new  type  of  experiment  would  add  evidence  to  one 
side  or  the  other  and  argue  for  or  against  auto-regeneration. 

While  there  could  be  found  no  other  microscopic  differences 
in  degeneration  in  medullated  fibers  after  section  and  in  medul- 


DEGENERATION  AND  REGENERATION  OF  NERVES       81 

lated  fibers  of  the  rice-fed  fowls  at  the  time  of  paralysis,  multi- 
plication of  the  nuclei  of  the  neurilemma  sheath  and  the  embry- 
onic nerve  fibers  were  conspicuous  by  their  absence  from  the 
nerves  of  the  latter  fowls.  Could  regeneration  be  accomphshed 
in  such  nerves,  without  a  multiplication  of  the  neurilemma  nuclei, 
the  significance  of  the  embryonic  nerve  fiber  would  be  minimized. 

With  the  above  questions  in  mind,  regeneration  was  studied 
in  the  sciatic  nerve  of  fowls  which  came  down  in  20  to  30  days 
with  marked  leg  paralysis  and  which  were,  from  time  to  time, 
placed  on  a  regeneration  diet.  To  recapitulate  briefly  certain 
observations  noted  above;  in  fowls  of  this  class,  those  medullated 
fibers,  presenting  at  the  time  of  paralysis  the  most  marked  degen- 
eration showed,  at  most,  only  a  doubtful  increase  in  the  number 
of  nuclei  of  the  neurilemma  sheath  and  no  embryonic  nerve 
fibers.  Nerves  of  fowls  killed  after  feeding  from  4  days  to  2 
months  on  the  regeneration  diet  never  showed  the  marked  multi- 
plication of  the  nuclei  of  the  neurilemma  sheath  or  the  embryonic 
nerve  fiber,  as  has  been  constantly  described  for  mammalian 
nerves  after  section,  and  as  was  found  also  in  the  nerves  of  fowls 
in  which  I  had  transsected  the  sciatic.  Segments  of  the  sciatic 
of  one  side  cut  out  and  compared  with  the  sciatic  of  the  other 
side  at  a  later  date,  showed  that  in  none  of  these  cases  had  the 
looked  for  change  in  this  respect  taken  place.  In  other  nerves 
after  108,  125,  171  and  275  days  in  regeneration,  the  nuclei  of 
the  sheath  of  Schwann  could  not  be  said  to  be  more  numerous 
than  in  preparations  taken  at  the  time  paralysis  developed,  and 
the  embryonic  nerve  fiber  could  not  be  found.  The  close  resem- 
blance of  the  Marchi  preparation  from  fowls  108  days  and  171 
days  on  a  regeneration  diet  to  those  from  fowls  at  the  time  of 
paralysis  has  also  been  pointed  out  and  is  clearly  seen  by  com- 
paring figures  2,  3,  4  and  5  with  figures  13  and  14.  Numerous 
large  globules  and  small  droplets  of  degenerated  myelin  are  to 
be  seen  in  each. 

In  view  of  this  condition  of  the  myelin,  the  uniform  absence 
of  embryonic  nerve  fibers,  and  the  fact  that  a  great  majority  of 
fibers  of  the  sciatic  do  not  show  a  breaking  up  of  the  axis  cylin- 
der at  the  time  of  paralysis,  it  was  at  first  suspected  that  regen- 

THE  JOURN.^L  OF  COMPARATIVE  NEDROLOGY,  VOL.  24,   XO.  1 


82  ELBERT   CLARK 

eration  failed  to  take  place  in  those  medullated  fibers  showing 
the  most  marked  degeneration;  and  that  in  its  recovery  the  fowl 
gradually  learned  to  do  without  these  fibers.  Further  study 
convinced  me,  however,  that  this  was  not  the  case,  and  that 
those  fibers  which  had  shown  such  marked  degeneration  finally 
attained  a  new  axis  cylinder.  This  conclusion  became  evident 
after  a  close  study  of  the  axis  cylinder  in  those  fowls  which  had 
recovered  from  paralysis. 

Mallory's  phosphomolybdic  acid  hematoxylin,  carmine,  Cajal's 
new  silver  impregnation  method  for  axis  cylinders,  and  Ranson's 
modification  of  the  last  were  used  for  staining  the  axis  cylinder. 
The  preliminary  treatment  (i.e.,  hardening  in  absolute  alcohol) 
called  for  in  the  silver  methods  produced  such  shrinkage  of  the 
fibers  that  it  was  often  impossible  to  obtain  satisfactory  teased 
preparations.    The  degenerated  myelin  was  also  dissolved  out 
to  such  an  extent,  that  together  with  the  shrinkage,  relations 
were  so  distorted  that  it  was  usually  difficult  to  distinguish  an 
old  from  a  new  axis  cylinder  and  to  tell  the  relation  of  the  latter 
to  the  globules  of  myelin.    With  the  first  two  methods  it  was 
possible  to  stain  a  preparation  in  bulk,  clear  and  tease  out  with- 
out passing  through  the  higher  alcohols  or  xylol.    The  globules 
of  degenerated  myelin  were  thus  preserved.    With  the  phospho- 
molybdic acid  hematoxylin,  which  gave  beautiful  results  after 
proper  fixation  with  Miiller's  fluid,  the  procedure  was  as  follows: 
Fix  in  Miiller's  fluid  (small  pieces  as  fresh  as  possible),  wash  1 
to  2  hours  in  running  water,  partially  tease  out  a  segment  of  the 
nerve  not  more  than  1  mm.  long  to  permit  rapid  infiltration  of 
the  stain,  place  in  a  ripened  solution  of  Mallory's  phosphomo- 
lybdic acid  hematoxylin  20  minutes  to  over  night,  blot  off  excess 
of  stain  and  differentiate  in  50  per  cent  alcohol  made  slightly 
alkaline  with  ammonia,  pass  through  two  or  three  changes  of  91 
per  cent  alcohol,  clear  in  origanum  oil,  tease,  blot,  and  mount 
in  xylol  balsam.     Carmine  preparations  were  prepared  in  a  simi- 
lar manner  but  differentiated  in  weak  alcohol  without  the  ammo- 
nia.    For  longitudinal  and  cross  sections,  pieces  of  nerve  after 
washing  were  ra]:)idly  dehydrated  and  cleared  and  mounted  in 
paraffin  and  stained  as  indicated.     Tissues  were  also   fixed  in 


DEGENERATION  AN^  REGENERATION  OF  NERVES       83 

alcohol  and  in  Bensley's  chrom-sublimate  solution  for  carmine 
staining. 

It  had  been  previously  shown  by  me  ('12),  that,  in  degener- 
ation in  the  rice-fed  fowls  at  time  of  paralysis,  there  is  just  as 
large  a  percentage  of  fibers  showing  advanced  degeneration  in 
the  sciatic  as  in  its  peripheral  rami.  In  regeneration,  then,  there 
should  be,  in  the  earlier  stages,  a  greater  percentage  of  fibers 
containing  no  axis  cylinder  in  the  peripheral  branches  than  in 
the  sciatic  itself;  provided  of  course,  the  new  axis  cyUnder  is 
the  result  of  an  outgrowth.  To  determine  this,  segments  of  the 
sciatic  and  its  peripheral  rami  were  cut  out  in  the  above  series 
of  regenerating  fowls  for  comparison  with  each  other  and  with 
the  sciatic  of  the  other  side  at  a  later  date.  • 

The  first  indication  of  a  regeneration  of  the  axis  cylinder  was 
obtained  from  fowl  No.  54  which  developed  marked  leg  paralysis 
on  March  4,  1912.  The  animal  was  placed  on  the  regeneration 
diet  on  March  7.  By  May  3,  all  signs  of  neuritis  had  disappeared 
and  complete  use  of  the  legs  had  been  regained.  On  this  day, 
on  the  left  side,  segments  of  the  nerve  were  cut  out  from  the 
upper  part  of  the  thigh,  and  from  near  the  foot.  An  examination 
of  transverse  sections  of  these  two  pieces  revealed  a  greater  pro- 
portion of  medullated  fibers  devoid  of  axis  cylinder  in  the  periph- 
eral segment  than  in  the  segment  from  the  thigh.  Out  of  742 
fibers  in  the  peripheral  portion,  the  axis  cylinder  was  wanting 
in  11;  in  1365  fibers  (counted  at  random)  in  the  proximal  por- 
tion, the  axis  cylinder  was  wanting  in  9.  The  fowl  was  killed 
114  days  later,  on  August  25.  Transverse  sections  of  the  sciatic 
of  the  opposite  side  on  this  date,  revealed  an  axis  cylinder  in 
practically  every  fiber.  In  5788  fibers  the  axis  cylinder  was 
wanting  in  8.  Similar  data  were  obtained  from  the  nerves  of 
other  rice-fed  fowls,  No.  52,  No.  57  and  No.  64.  However,  as 
degenerated  fibers  may  not  be  evenly  distributed  throughout- 
the  sciatic,  one  peripheral  nerve  may  contain  a  larger  percentage 
of  degenerated  fibers  than  its  neighbor  and  relatively  more  than 
the  sciatic  of  which  it  is  a  branch.  As  misleading  results  might 
thus  be  obtained,  this  method  of  comparison  was  not  prosecuted 
further. 


84  ELBERT   CLARK 

These  findings  having  indicated  that,  under  favorable  condi- 
tions, regeneration  of  the  axis  cyhnder  may  take  place  in  the 
degenerated  nerves  of  the  rice-fed  fowls,  more  definite  evidence 
of  regeneration  was  sought.  This  was  a  difficult  and  tedious 
task  because  it  was  necessary  to  show  beyond  doubt  that  an  axis 
cylinder  in  a  particular  nerve  was  a  new  and  not  an  old  axis 
cylinder.  Therefore  a  most  careful  search  was  made  of  sectioned 
and  teased  preparations  of  nerves  taken  at  varying  lengths  of 
time  after  recovery  from  paralysis. 

The  sciatic  of  fowl  No.  38 — 108  days  after  regeneration  diet 
was  begun  and  49  days  after  paralysis  had  disappeared — was 
particularly  studied  because,  in  addition  to  being  a  typical  case 
of  peripheral  neuritis,  the  axis  cylinders  stained  exceptionally 
well  and  many  globules  and  droplets  of  myelin  were  shown  by 
the  Marchi  stain  (fig.  13).  In  a  large  fiber  containing  several 
large  globules  of  degenerated  myelin  along  its  course,  a  well  stain- 
ing axis  cylinder  was  seen  running  a  tortuous  course  to  one 
side  of  the  large  globules  which  often  occupied  almost  the  entire 
diameter  of  the  fiber.  Two  such  vesicular  globules  of  degen- 
erated myelin  in  close  proximity  to  the  axis  cylinder  were  seen, 
which  contained  in  their  center  segments  of  a  structure  which 
was  identified  as  the  old  axis  cylinder.  In  figure  17,  m  shows 
one  of  the  globules  in  question  with  its  axis  cylinder  contents. 
In  this  figure,  m  clearly  represents  a  single  globule  of  degenerated 
myelin  which  has  been  cut  on  the  tangent  by  the  microtome 
knife.  Part  of  the  old  axis  cylinder  was  also  probably  taken 
away.  The  different  portions  of  a  and  a' ,  the  new  and  the  old 
axis  cylinders,  were  not  in  focus  at  the  same  time  and  the  draw- 
ing has  been  constructed,  with  the  aid  of  a  camera  lucida  outline, 
to  show  as  nearly  as  possible  in  one  plane,  the  relations  of  these 
structures.  In  this  figure,  a'  is  the  exact  counterpart  of  a  in  figure 
6  which  is  readily  recognized  as  broken  up  axis  cylinders  within 
large  globules  of  degenerated  myelin.  That  a,  figure  17,  repre- 
sents an  axis  cylinder  there  can  be  no  doubt. 

This  observation  has  been  confirmed  in  other  fibers  of  this 
same  nerve,  as  well  as  in  the  fibers  of  the  sciatic  of  fowl  No. 
51,  No.  54,  and  No.  61.     In  each  of  these  there  could  be  no 


DEGENERATION  AND  REGENERATION  OF  NERVES       85 

doubt  about  the  identity  of  the  structures.  When  it  is  remem- 
bered that  the  axis  cyhnder  in  advanced  degeneration  is  often 
quite  difficult  to  stain,  it  is  not  surprising  that  such  clear  pic- 
tures as  the  above  (fig.  17)  were  not  frequently  found.  The 
tortuous  course  of  the  new  axis  cylinder  around  the  globules, 
as  well  as  the  different  focal  levels  of  fragments  of  the  old,  makes 
it  extremely  difficult  to  get  a  photomicrographic  representation 
which  will  show  both  structures  in  one  picture.  Figure  18  shows 
a  fragment  of  the  old  axis  cylinder,  a' ,  inclosed  within  a  large 
globule  of  degenerated  myelin,  m,  in  close  proximity  to  the  new 
axis  cylinder  a.  In  figures  19  and  20  the  same  condition  is 
shown:  a',  the  fragment  of  the  old  axis  cylinder;  m,  a  globule  of 
degenerated  myelin  and  a,  the  new  axis  cyhnder.  It  is  more 
frequently  the  case  that  the  remains  of  the  old  axis  cylinder 
are  represented  only  by  a  mass  of  granules  or  fragments  enclosed 
within  the  globule. 

Further  confirmation  of  these  observations  was  readily  ob- 
tained by  a  study  of  cross-sections  of  the  sciatic  of  these  same 
fowls.  In  such  sections  the  new  axis  cylinder  could  be  seen  at 
one  side  of  the  myelin  globules  while  segments  or  fragments  of 
the  old  were  to  be  seen  within  the  globule.  In  some  fibers  the 
new  axis  cylinder  was  a  very  small  structure,  0.5ai  or  less  in 
diameter,  and  located  quite  at  the  periphery  of  the  sheath.  In 
other  fibers  it  was  larger  and  with  its  surrounding  concentric 
lamellae  occupied  an  equal  proportion  of  the  sheath  with  the 
globules  of  degenerated  myelin.  Figure  21,  fowl  No.  54,  shows 
a  new  axis  cylinder,  a,  the  old  axis  cylinder,  a',  and  a  globule  of 
degenerated  myehn,  m,  in  the  same  cross-section.  Figure  22 — 
fowl  No.  38 — shows  a  large  new  axis  cyhnder,  a,  with  its  con- 
centric lamellae,  s,  by  the  side  of  the  old  axial  tube,  a;  a'  \s,  also 
surrounded  by  concentric  lamellae  and  no  large  globules  of 
myelin  are  seen  here.  It  is  probable  that  this  is  a  section  of  a 
nerve  devoid  of  myelin  globules  at  this  place  and  in  which  the 
axis  cylinder  has  degenerated  as  a  result  of  its  interruption  by 
myelin  globules  at  another  level.  Figures  23  and  24  are  photo- 
micrographs respectively  of  the  same  preparations  as  figures  21 
and  22. 


86  ELBERT    CLARK 

All  these  observations  speak  strongly  for  a  new  axis  cylinder 
in  recovery;  the  greater  percentage  of  fibers  with  axis  cylinders 
in  the  sciatic  than  in  its  peripheral  rami  argues  also  for  an  out- 
growth of  the  axis  cylinder.  Further  evidence  of  outgrowth  of 
the  axis  cjdinder  was  soon  obtained.  Before  I  was  able  to  con- 
firm the  first  observation  that  a  new  axis  cylinder  and  segments 
of  the  old  were  to  be  found  in  the  same  fiber  at  the  same  time, 
another  fiber  was  observed  in  the  same  preparation,  in  which 
growth  activity  was  apparent.  This  fiber  is  shown  in  figures 
27  and  figures  28  and  29  (photomicrographs)  all  of  the  same  fiber. 
It  will  readily  be  seen  that  6  is  an  outgrowing  branch  of  the 
axis  cylinder  a.  That  a  is  a  new  axis  cylinder  is  proven  by  the 
presence  of  a  fragment  of  the  old  axis  cylinder,  a',  (fig.  27  and 
fig.  29)  between  the  new,  a,  and  its  branch,  b.  Both  axis  cylinder 
and  branch  stained  equally  well  and  much  better  than  the  rem- 
nants of  the  old.  An  end  bulb  is  seen  on  the  tip  of  the  branch. 
It  might  be  pointed  out  here  that  Cajal  and  Marienesco  have 
observed  a  similar  branching  of  the  outgrowing  axis  cylinder  in 
medullated  nerves  after  section.  These  branches,  of  which  there 
may  be  one  or  more  to  each  fiber,  often  take  an  abortive  course 
and  have  been  observed  to  grow  in  a  recurrent  direction  up  the 
central  stump.  Whether,  in  the  present  case,  b  is  an  outgrowth 
from  a  or  whether  at  an  earher  stage  both  were  outgrowing  buds 
of  approximately  equal  size  is  purely  a  matter  of  speculation. 
It  should  be  added  that  in  cross-sections  old  sheaths  were  ob- 
served which  contained  two  axis  cyhnders  of  approximately  equal 
size,  each  surrounded  by  a  secondary  sheath  of  its  own.  But 
more  frequently  one  is  much  larger  and  occupies  a  more  cen- 
tral position  than  the  smaller  which  may  be  located  quite  near 
the  periphery  of  the  sheath.  Figures  25  and  26  from  the  sciatic 
of  fowl  No.  54,  show  in  transverse  section  two  axis  cylinders  in 
the  same  nerve  sheath,  and  apparently  in  the  same  portion  of 
the  fiber  that  was  formerly  occupied  by  the  old  axis  cyUnder. 
WTiether  the  zone  around  each  represents  a  newly  acquired  mye- 
lin sheath  I  have  not  determined.  Nuclei,  however,  have  not 
been  observed  in  this  zone. 


DEGENERATION  AND  REGENERATION  OF  NERVES       87 

Other  than  the  rami  as  just  noted  (fig.  27),  an  outgrowing 
axis  cylinder  with  its  end  bulb,  as  described  by  Cajal  ('07),  has 
been  observed  only  once  by  me.  Figure  30 — ^from  a  teased  prep- 
aration of  the  sciatic  nerve  of  No.  38 — shows  in  a,  a  structure 
with  an  end  bulb  which  stained  intensely  with  phosphomolybdic 
acid  hematoxylin  and  which  is  lodged  in  a  band  of  poorly  stain- 
ing tissue  rich  in  nuclei.  Whether  the  band  of  tissue  is  a  group 
of  embryonic  nerve  fibers  or  non-medullated  fibers  in  regener- 
ation, I  cannot  say. 

In  the  light  of  this  group  of  evidence  which  bespeaks  an  out- 
growth of  the  axis  cylinder  into  the  old  nerve  fiber  sheath,  cer- 
tain observations  of  Cajal  ('07),  Marienesco  ('06),  Ranson  ('12) 
and  others  gain  an  additional  interest.  These  investigators  found 
that,  almost  immediately  after  section,  the  axis  cylinders  of  the 
central  stump  showed  evidence  of  growth  activity.  ''Marien- 
esco, in  one  of  his  recently  published  papers  has  demonstrated 
that  the  letigthening  of  the  regenerating  fibers  (i.e.,  axis  cyhnders) 
is  demonstrable  twenty-four  hours  after  a  nerve  has  been  cut" 
(Halliburton).     Ranson  ('12)  says: 

On  the  first  day  after  the  lesion  some  of  the  axons  grow  out  into  the 
exudate  and  break  up  into  many  branches  (fig.  16).  Others  on  the 
first  day,  give  off  fine  branches  from  their  surface  within  the  sheath 
in  the  immediate  neighborhood  of  the  lesion  (fig.  17),  some  of  which 
find  their  way  into  the  exudate.  Thus  from  the  end  of  the  first  day 
on,  fine  nerve  fibers,  which  are  demonstrably  branches  of  the  medullated 
axons  of  the  proximal  stump,  are  present  in  the  developing  scar,  and 
running  for  the  most  part  within  the  sheath  of  the  old 
axon  from  which  they  arose,  they  arrange  themselves  into  fascicles,  etc. 

Cajal's  beautiful  figures  illustrate  most  clearly  the  axis  cyhn- 
der  growing  down  into  the  old  sheath  of  that  portion  of  the 
central  stump  which  showed  degeneration  after  section.  Howell 
and  Huber  ('92)  also  observed  a-  similar  growth  of  the  axis  cylin- 
der in  the  central  stump.  Branches  of  these  axis  cylinders  are 
also  to  be  seen  growing  up  the  medullated  fiber  in  a  central 
direction;  others  burst  through  the  sheath  into  the  interfibrillar 
tissue,  and  still  others  after  invading  the  blood  clot  and  inflam- 
matory tissue  between  the  sutured  central  and  peripheral  stumps 


88  ELBERT   CLARK 

are  seen  to  grow  down  into  the  old  fiber  sheaths  of  the  peripheral 
stump.  All  this  may  take  place  before  there  is  any  marked  in- 
crease in  the  number  of  nuclei  of  the  neurilemma  sheath  and 
long  before  embrj^onic  nerve  fibers  develop.  It  is  further  a  com- 
mon observation  (as  pointed  out  by  Langley  and  Anderson  ('04)) 
that,  unless  very  special  precautions  are  taken  to  prevent  it, 
the  peripheral  stump  is  invaded  by  foreign  fibers  from  neigh- 
boring nerves;  and  Forsmanns  has  shown  that  even  macerated 
brain  tissue  exerts  a  chemotactic  influence  on  the  outgrowing 
axis  cylinders. 

Clearly  then  the  importance  per  se  of  the  embryonic  nerve 
fiber  in  the  regeneration  of  medullated  nerves  has  been  greatly 
overestimated. 

To  summarize  briefly,  there  have  been  observed  in  the  nerves 
of  this  series  of  fowls  which  have  recovered  from  a  pronounced 
paralysis  of  the  legs  brought  on  by  a  prolonged  diet  of  white 
rice,  the  following:  sections  of  nerves,  taken  from  the  sciatic  and 
its  peripheral  branches  at  various  times  during  recovery  of  the 
fowl,  showed  a  greater  percentage  of  fibers  possessing  axis  cylin- 
ders in  the  sciatic  than  in  its  peripheral  branches.  In  regener- 
ation, a  new  axis  cylinder  was  acquired  by  those  nerve  fibers  in 
which  a  long  series  of  observations  prove  that  the  axis  cylinder 
and  myelin  sheath  had  undergone  marked  degeneration.  The 
large  globules  and  small  droplets  of  degenerated  myelin  persisted 
several  months  after  complete  recovery  of  the  fowl.  Multipli- 
cation of  the  nuclei  of  the  neurilemma  sheath  and  the  resulting 
embryonic  nerve  fiber  were  lacking  or  were  of  the  greatest  infre- 
quency  in  regenerating  as  well  as  degenerating  medullated  fibers. 
A  new  axis  cylinder  and  segments  or  fragments  of  the  old  and 
globules  of  degenerated  myelin  were  found  together  in  the  same 
nerve  fiber,  whose  neurilemma  sheath  showed  no  increase  in  its 
nuclei.  A  new  axis  cylinder,  a  branch  of  the  same  ending  in 
a  bulb,  fragments  of  the  old  axis  cylinder  and  globules  of  degen- 
erated myelin  (and  with  no  multiplication  of  the  nuclei  of  the 
neurilemma  sheath)  were  all  seen  in  the  same  portion  of  a  regen- 
erating fiber.  Two  axis  cylinders  in  the  same  fiber  and  indica- 
tions of  an  outgrowing  axis  cylinder  were  observed. 


DEGENERATION  AND  REGENERATION  OF  NERVES       89 

From  these  facts  it  is  clear  that  neither  the  nuclei  of  the  sheath 
of  Schwann  nor  the  embryonic  nerve  fiber  could  have  taken 
any  part  in  the  formation  of  the  new  axis  cyUnder.  Conse- 
quently auto-regeneration  in  so  far  as  it  signifies  the  formation 
of  a  new  axis  cylinder  by  the  embryonic  nerve  fiber  does  not 
obtain  with  fowls  in  regeneration  after  paralysis  from  polished 
rice. 

The  same  observations  which  show  that  the  new  axis  cylinder, 
in  these  experiments,  is  not  acquired  through  auto-regeneration, 
also  demonstrate  that  it  is  attained  by  outgrowth.  The  pres- 
ence of  a  new  axis  cyhnder  and  segments  of  the  old  in  the  same 
old  medullary  sheath;  the  presence  of  two  new  axis  cyhnders  in 
an  old  sheath,  and  the  occurrence  of  a  new  axis  cylinder  with 
an  outgrowing  branch,  and  of  an  outgrowing  axis  cylinder  with 
an  end  bulb,  can  only  mean  that,  in  the  absence  of  auto-regen- 
eration, the  new  axis  cylinder  has  grown  out  from  its  central 
stump.  ^ 

Regeneration  in  the  cord 

Before  considering  the  possibility  of  regeneration  in  the  fibers 
of  the  cord,  it  is  necessary  to  refer  to  the  degenerative  changes 
in  the  meduUated  fibers  and  nerve  cells  of  the  cord  described  by 
me  in  a  recent  study  of  ''Polyneuritis  Gallinarum"  ('12).  Here 
it  was  found  that  a  very  small  per  cent  of  the  fibers  of  all  columns 
of  the  cord  presented  as  advanced  myehn  degeneration,  as  the 
fibers  of  the  peripheral  nerves.  A  still  smaller  per  cent  of  the 
fibers  also  showed  a  disintegration  or  breaking  up  of  the  axis 
cylinder.     The  nerve  cells  of  the  ventral  horn  and  the  basal 

^  The  question  naturally  arises,  at  what  point  does  this  outgrowth  of  the  axis 
cylinder  begin?  I  have  not  been  able  to  answer  this  satisfactorily.  One  would 
suppose  that  outgrowth  would  begin  at  the  peripheral  end  of  that  segment  of 
the  axis  cylinder  (still  connected  with  the  nerve  cell)  which  did  not  undergo 
degeneration;  if  such  a  segment  exists.  As  stated  below,  I  have  not  been  able 
to  determine  if  that  portion  of  the  axis  cylinder  running  between  the  anterior 
horn  cells  and  the  periphery  of  the  cord  ever  shows  segmentation,  such  segmen- 
tation not  having  been  observed.  Both  ventral  and  dorsal  nerve  roots,  on  the 
other  hand,  have  been  frequently  observed  in  which  segmentation  and  disinte- 
gration of  the  axis  cylinder  and  clumping  of  the  myelin  were  clearly  visible. 


90  ELBERT   CLARK 

portion  of  the  dorsal  horn  experienced  marked  changes  in  their 
chromophile,  Nissl,  or  tigroid  substance,  the  globules  or  flocculi 
had  given  way  to  a  uniform,  finely  granular  mass  collected  at 
one  side  of  the  nerve  cell,  usually  at  the  base  of  one  of  the  proc- 
esses of  the  cell.  I  was  not  able  to  determine  whether  that 
portion  of  the  axone  between  the  motor  nerve  cell  and  the  pe- 
riphery of  the  cord  ever  underwent  segmentation. 

A  close  examination  of  sections  of  the  cords  of  Nos.  38,  57, 
61  and  64  has  revealed  a  persistence  of  globules  of  degenerated 
myelin  in  the  medullated  fibers  of  all  columns  of  the  cord  for 
59,  108,  275  and  379  days.  These  globules  were  frequently  very 
large  and  occupied  the  entire  diameter  of  the  fiber.  Further- 
more, a  careful  search  has  failed  to  reveal  any  evidence  of  regen- 
eration in  the  fibers  of  the  cord.  No  such  proof  of  regeneration 
as  was  found  in  the  fibers  of  the  sciatic  and  illustrated  in  figures 
17  to  30  was  found  in  the  cord.  Nothing  suggestive  of  a  new 
axis  cylinder,  an  outgrowth  or  branching  of  the  same  was  seen 
and  there  were  not  observed  two  axis  cylinders  in  the  same 
fiber. 

On  the  other  hand,  fibers  in  degeneration  in  all  columns  of 
the  cord  were  found  in  which  no  new  axis  cylinder  nor  fragments  of 
the  old  were  observed.  Figure  31  is  a  cross-section  of  a  degen- 
erated fiber  49  days  after  complete  recovery  of  fowl  No.  38 
(108  days  in  regeneration)  was  attained.  A  large  globule  of 
degenerated  myelin  completely  fills  up  and  distends  the  sheath 
and  no  indication  of  the  axis  cylinder  is  to  be  seen. 

On  comparing  cross-sections  of  the  lumbo-sacral  cord  of  fowls 
taken  at  the  time  paralysis  developed  with  sections  from  the 
same  region  of  other  fowls  several  months  after  complete  recov- 
ery, the  data  included  in  table  1  were  obtained. 

From  this  table  it  will  be  seen  that  there  are  as  many  degen- 
erated fibers  with  no  axis  cylinder  in  the  cords  of  those  fowls 
killed  several  months  after  recovery,  as  in  the  cords  of  fowls 
killed  at  the  time  paralysis  developed. 

In  the  absence  of  any  positive  evidence  of  regeneration,  the 
persistence  of  degenerated  fibers  with  no  axis  cylinder,  in  as  great 
numbers  as  at  the  height  of  degeneration,  strongly  suggests  the 


DEGENERATION  AND  REGENERATION  OF  NERVES 


91 


conclusion  that  regeneration  in  the  medullated  fibers  of  the  cord 
of  the  rice-fed  fowls  fails  to  take  place. 

Regeneration  in  the  cord  was  included  in  this  study  because, 
after  regeneration  had  been  shown  to  take  place  in  the  peripheral 
nerves  by  an  outgrowth  of  the  axis  cylinder  into  the  old  medul- 
lary sheath  and  in  the  absence  of  the  embryonic  nerve  fiber,  no 
reason  was  now  apparent  why  the  same  thing  could  not  occur 
in  the  fibers  of  the  cord  as  well.  According  to  the  majority  of 
investigators,  regeneration  in  the  fibers  of  the  cord  is,  to  say  the 
most,  doubtful.  Schafer  ('08)  declares  "regeneration  does  not 
occur  within  the  central  nervous  system,  or  at  most  in  a  very 
incomplete  manner.  This  fact  may  be  associated  with  the  cir- 
cumstance that  the  fibers  within  the  spinal  cord  and  brain  have 
no  nucleated  sheath  of  Schwann,  and  the  conducting  path  which 
the  cells  of  this  sheath  form  in  the  peripheral  nerves  for  the  out- 
growing axis  cylinders  is  therefore  absent."  'According  to  Halli- 
burton ('07),  as  noted  above,  the  neurilemma  is  'non-existent' 
in  the  medullated  fibers  of  the  central  nervous  system  and  ''as 
is  well  known,  regeneration  does  not  occur"  in  these  fibers.     But 


TABLE  1 

Showing  degeneration  in  the  lumbosacral  cord  at  time  of  paralysis  and  after 

recovery 


NTJMBER  OF  DEGENEBATED  FIBERS 

WITH  NO  AXIS  CYLINDER  IN  THE  CORD 

FOWL  NO. 

OF  THE  FOWL  KILLED 

REMARKS 

At  time  of 

After  complete 

paralysia 

recovery 

14 

.   77 

,    1 

65 

13 

28 

38 

/59\ 

108  days  in  regeneration 

\67/ 

49  days  after  recovery 

57 

62 

379  days  in  regeneration 
300  days  after  recovery 

64 

57 

275  days    in  regeneration 
*256  days  after  recovery 

61 

• 

1171 

59  days  in  regeneration; 
no  recovery 

^  The  cord  of  this  fowl  also  contained  a  great  many  fibers  which  appeared  con- 
siderably swollen  and  which  are  not  included  in  this  count. 


92  ELBERT   CLARK 

as  noted,  if  regeneration  in  the  peripheral  nerves  was  accom- 
plished without  the  embryonic  nerve  fibers,  then  clearly  in  these 
fowls  the  absence  from  the  cord  of  the  neurilenmaa  sheath  and 
its  derivative,  the  embryonic  nerve  fiber,  would  afford  no  expla- 
nation of  a  failure  or  regeneration  in  the  fibers  of  the  cord,  and 
an  outgrowth  of  the  axis  cylinder  in  the  fibers  of  the  cord  might 
well  be  expected.  Such,  however,  as  the  evidence  shows,  is 
probably  never  the  case. 

Although  at  the  time  of  paralj^sis  the  marked  changes  described 
above  were  seen  in  the  nerve  cells  of  the  lumbo-sacral  cord,  it 
is  doubtful  that  this  should  be  termed  degeneration.  The  mito- 
chondria seem  to  have  undergone  no  alteration  whatsoever.  They 
were  just  as  numerous  as  in  the  nerve  cells  of  the  cord  of  the 
normal  fowl.  In  the  cells  of  the  cord  of  fowl  79,  killed  as  soon 
as  all  signs  of  paralysis  had  disappeared  (after  30  days  on  a 
special  regenerative  diet),  the  tigroid  bodies  again  presented  an 
appearance  similar  to  the  normal.  This  was  also  true  for  fowl 
No.  57  (10  months  after  complete  recovery).  No  'shadows'  or 
other  evidences  of  degenerated  nerve  cells  were  found  in  either 
case. 

GENERAL  SUMMARY 

In  the  experiments  described  above  degeneration  of  medul- 
lated  nerve  fibers  was  brought  about  in  fowls  by  a  prolonged 
feeding  of  polished  rice,  and  regeneration  was  accomplished  by 
a  return  to  an  adequate  nutritive  diet. 

In  such  fowls  the  fibers  are  intact  during  degeneration  and  all 
traumatic  and  inflammatory  effect  produced  by  cutting  the  tis- 
sues and  the  nerve  or  of  tying  the  latter  are  obviated ;  the  process 
of  degeneration  can  be  stopped  at  almost  any  stage  or  greatly 
prolonged,  and  several  stages  of  degeneration  are  to  be  observed 
in  different  fibers  of  the  same  nerve.  In  regeneration  the  possi- 
bility of  an  ingrowth  of  fibers  from  other  nerves  into  the  regen- 
erating nerve  under  observation  is  eliminated  and  repair  of  the 
medullated  nerves  can  be  induced  after  any  stage  of  degeneration. 

Ten  to  20  per  cent  of  the  medullated  fibers  of  the  nervus 
ischiadicus  showed  a  complete  fatty  change  of  their  medullary 


DEGENERATION  AND  REGENERATION  OF  NERVES       93 

sheaths  into  globules  of  degenerated  myelin  and  a  segmentation 
or  granulation  of  their  axis  cylinders.  No  multiplication  of  the 
nuclei  of  the  neurilemma  sheath  could  be  observed  and  conse- 
quently no  embryonic  nerve  fibers  or  Band-fasern. 

During  recovery  these  degenerated  fibers  attained  new  axis 
cylinders  and  the  medullary  sheaths  returned  to  normal.  In 
other  words,  regeneration  has  been  observed  to  follow  degener- 
ation in  meduUated  nerve  fibers  without  passing  through  the 
embryonic  nerve  fiber  or  Band-faser  stage. 

By  prolonging  the  degenerative  process  there  resulted  a  multi- 
plication of  the  nuclei  of  the  neurilemma  sheath.  This  and  other 
experiments  described  tend  to  show  that  the  embryonic  nerve 
fiber  may  be  coincident  with  a  late  stage  of  degeneration  in 
medullated  nerve  fibers.  It  may  not  represent  an  early  stage 
of  regeneration  and  its  presence  does  not  signify  an  attempt  at 
regeneration  on  the  part  of  the  medullated  nerve  fiber. 

In  the  absence  of  the  embryonic  nerve  fiber,  the  degenerated 
myehn  was  absorbed  with  extreme  slowness,  persisting  as  drop- 
lets after  1  year  and  14  days.  On  the  other  hand,  where  the 
embryonic  nerve  fiber  was  formed  the  degenerated  myelin  quickly 
disappeared  from  the  fiber.  The  conclusion  is  reached  that  the 
proliferating  nuclei  of  the  neurilemma  sheath  participate  in  the 
resorption  of  the  degenerated  myehn. 

In  regeneration  a  new  axis  cylinder  was  attained  by  outgrowth 
and  in  the  absence  of  the  embryonic  nerve  fiber.  The  new  axis 
cylinder  grew  down  the  old  medullary  sheath  which  latter  still 
contained  large  globules  of  degenerated  myelin  and  fragments 
of  the  old  axis  cylinder.  The  outgrowing  axis  cylinder  was  seen 
to  branch,  and  in  cross-sections  of  the  nerves  two  new  axis  cylin- 
ders were  observed  within  the  same  old  medullary  sheath.  The 
embryonic  nerve  fiber  could,  of  course,  play  no  part  in  the  for- 
mation of  the  new  axis  cylinder  either  by  auto-regeneration  or 
by  outgrowth. 

No  indications  of  regeneration  were  observed  in  the  fibers  of 
the  spinal  cord. 

Anatomical  Laboratory,  The  University  of  Chicago, 
June  28,  1913. 


94  ELBERT   CLARK 

BIBLIOGRAPHY- 

Ballance,  C.  a.,  and  Stewart,  P.  1901  The  healing  of  nerves;  London  (cited 
after  Mott,  Halliburton  and  Edmonds). 

Bethe,  Albrecht    1903    Neurol.  Centralbl.    Bd.  22,  January,  p.  60. 

1907  NeueVersucheiiber  die  Regeneration  der  Nervenfasern.  Archiv 
f.  d.  ges.  Physiol.,  Bd.  116,  p.  385. 

Cajal,  Ram6n'  y  1906  Mecanisme  de  la  regeneration  du  nerf.  Compt.  rend. 
Soc.  de  Biol.,  vol.  59,  p.  420. 

1905  and  1907  Mecanismo  de  la  regeneraci^n  de  los  nervi6s.  Trabajos 
del  laboratio  de  investigaciones  biologicas  de  la  Universidad  de  Madrid. 
(Illustrations  shown  by  Schafer  in  Quain's  anatomy). 

EiJKMAN,  C.  1897  Eine  Beri  Beri-ahnliche  Krankheit  der  Hiihner.  Virchow's 
Arch.,  Bd.  148,  p.  523. 

Frazer,  Henry,  and  Stanton,  A.  T.  1911  The  etiology  of  beri-beri.  Studies 
from  the  Institute  for  Medical  Research,  Federated  Malay  States, 
no.  12. 

Halliburton,  W.  D.  1907  Nervous  degeneration  and  regeneration.  Brit.  Med. 
Jour.,  vol.  1,  pp.  1111  and  1460. 

Howell,  W.  H.,  and  Huber,  G.  C.  1892  A  physiological,  histological  and 
clinical  study  of  the  degeneration  and  regeneration  in  peripheral  nerve 
fibers  after  severance  of  their  connections  with  the  nerve  centers. 
Jour.  Physiol.,  vol.  13,  p.  335. 

Hxjber,  G.  C.  1895  A  study  of  the  operative  treatment  for  loss  of  nerve  sub- 
stance in  peripheral  nerves.  Jour.  Morph.,  vol.  11,  p.  629  (cited  by 
Ranson). 

Langley,  J.  N.,  and  Anderson,  H.  K.  1904  On  autogenetic  regeneration  in  the 
nerves  of  the  limbs.     Jour.  Physiol.,  vol.  31,  p.  418. 

LuGARO,  E.     1906    Neurol.  Centralbl.,  Bd.  25,  p.  786  (cited  after  Ranson). 

-Marienesco,  G.  1906  Jour.  f.  Psychol,  u.  Neurol.,  Bd.  7,  p.  141  (cited  after 
Halliburton). 

Mott,  F.  W.,  Halliburton,  W.  D.,  and  Edmonds,  Arthur  1904  Regeneration 
of  nerves.    Jour.  Physiol.,  vol.  31,  p.  vii. 

1906  Regeneration  of  nerves.     Proc.  Roy.  Soc.  B.,  vol.  78,  p.  259. 

Nageotte,  J.  1911  Betrachtungen  liber  der  tatsachlichen  Bau  und  die  kiinst- 
lich  hervorgerufenen  Deformationen  der  markhatigen  Nervenfaser. 
Archiv  f.  mik.  Anat.,  Bd.  77,  Abt.  1,  p.  245. 


^  This  list  of  references  is  meant  to  include  only  such  of  the  available  literature 
as  has  a  direct  bearing  on  the  subjects  discussed. 


DEGENERATION  AND  REGENERATION  OF  NERVES       95 

Nageotte,  J.  1911  R61e  des  corps  granuleux  dans  la  phagocytose  du  neurite, 
au  cours  de  la  d^g6n6ration  Wall^rienne.  Comp.  rendu.  Soc.  de  Biol., 
vol.  71,  p.  251. 

Ranson,  S.  Walter  1912  Degeneration  and  regeneration  of  nerve  fibers.  Jour. 
Comp.  Neur.,  vol.  22,  p.  487. 

Schafer,  E.  a.  Quain's  eleinents  of  anatomy,  eleventh  edition,  p.  41.  New 
York  and  London. 

Schroder,  Paul  1908  Einfuhrung  in  die  Histologie  und  Histopathologie  des 
Nervensystems.    Jena. 

Stroebe,  H.  1893  Experimentelle  UnterSuchungen  liber  Degeneration  and  Re- 
generation peripherer  Nerven  nach  Verletzungen.  Beitr.  z.  path. 
Anat.  u.  z.  allegem.  Pathol.,  Bd.  13,  p.  160. 

Van  Gehuchten,  A.  1904  Bull,  de  I'Acad,  Roy.  de  Med.  de  Belgique,  p.  50 
(cited  after  Langley  and  Anderson). 

Vedder,  Edward  B.,  and  Clark,  Elbert  1912  A  study  of  polyneuritis  galli- 
narum.     Phil.  Jour.  Science,  vol.  7,  sec.  B,  p.  423. 

Wilson,  J.  Gordon  1909  The  present  position  of  the  theory  of  auto-regener- 
ation of  nerves.    Anat.  Rec,  vol.  3,  p.  27. 


PLATE  18 

EXPLANATION  OF  FIGURES 

1  Photomicrograph  of  a  teased  preparation  of  the  nervus  ischiadicus  of  a 
normal  fowl.     Marchi  method.     Zeiss  4  X  16  mm. 

2  Photomicrograph  of  a  teased  preparation  of  the  nervus  ischiadicus  of 
fowl  No.  2,  after  24  days  on  an  exclusive  diet  of  polished  rice.  Every  fiber  shows 
degeneration.  Advanced  degeneration  is  seen  in  4  fibers.  Marchi  method. 
Ziess  4  X  16  mm. 

3  Fiber  from  the  nervus  ischiadicus  of  fowl  No.  6 — 3  days  in  paralysis — 
showing  marked  degenerative  changes,  a  swelling  of  the  fiber  at  a,  b,  etc.,  and 
no  nuclei  of  the  neurilemma  sheath,  m  is  a  hollow  globule  of  degenerated  myelin 
containing  a  segment  of  the  axis  cylinder.     Marchi  method.     X  200. 

4  Fiber  from  the  nervus  ischiadicus  of  fowl  No.  1 — showing  complete  altera- 
tion of  the  medullary  sheath  into  globules  of  degenerated  myelin.  The  laminated 
appearance  of  the  larger  globules  is  seen  at  m.  n  and  n'  are  nuclei  of  the  neu- 
rilemma sheath.     Marchi  method.     X  312. 


*  Camera  lucida  outlines  were  used  in  the  preparation  of  all  the  drawings. 
The  photomicrographs  are  by  Martin  and  Cortes. 

96 


DEGENERATION  AND  REGENERATION  OF  NERVES 

ELBERT  CLARK 


PLATE 


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THE  JOURNAL  OP  COMPARATIVE  NEUROLOGY,  VOL.  24,  NO.  1 


PLATE  2 

EXPLANATION  OF  FIGURES 

5  Fiber  of  the  nervus  ischiadicus  of  fowl  No.  6.  No  multiplication  of  nuclei 
of  the  neurilemma  sheath  is  seen.     Marchi  method.     Zeiss  4X4  mm. 

6  Photomicrograph  of  section  of  nervus  ischiadicus  of  fowl  No.  9,  G — 52 
days  on  polished  rice  and  calcium  lactate,  a,  a'  segments  of  discontinuous  axis 
cylinders  enclosed  within  enlarged  portions  (globules  of  degenerated  myelin)  of 
the  fiber.  Mallory's  phosphomolybdic  acid  hematoxylin.  Zeiss  4X2  mm.  oil 
immersion. 

7  Photomicrograph  of  teased  preparation  of  nervus  ischiadicus  of  fowl  No.  6 
— 24  days  on  polished  rice.  Several  stages  of  degeneration  are  shown.  Fiber, 
a,  showing  advanced  degeneration  lies  side  by  side  with  one  showing  only  slight 
degenerative  change.     Marchi  method.     Zeiss  4X4  mm. 


DEGENERATION  AND  REGENERATION  OF  NERVES 

ELBERT   CLARK 


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EXPLANATION  OP  FIGURES 

8  Photomicrograph  of  teased  preparation  of  nervus  ischiadicus  of  fowl  No. 
78 — dry  gangrene  in  left  leg  which  had  been  bandaged  for  24  hours  28  days  be- 
fore death.  Slender  embryonic  nerve  fibers  with  numerous  spindle-shaped  nuclei 
have  entirely  replaced  the  meduUated  fibers.  Delafield's  hematoxylin,  Zeiss 
4X2  mm.  oil  immersion. 

9  Photomicrograph  of  embryonic  nerve  fibers  in  the  nervus  ischiadicus  of 
fowl  No.  17,  G — 60  days  on  polished  rice  and  calcium  lactate.  Delafield's  hema- 
toxylin.    Zeiss  4X2  mm.  oil  immersion. 

10  Embryonic  nerve  fiber  from  the  same  nerve  as  figure  9.  Droplets  of 
degenerated  myelin  are  seen  at  m.    Delafield's  hematoxylin.     X  255. 

11  Embryoni  •  nerve  fiber  from  a  teased  preparation  of  the  nervus  ischi- 
adicus of  fowl  No.  9,  G — ^52  days  on  polished  rice  and  calcium  lactate;  droplets 
of  degenerated  myelin  are  seen  at/;  n,  nucleus.     Delafield's  hematoxylin.     X  250. 

12  Fiber  of  the  nervus  ischiadicus  of  fowl  No.  14,  showing  infiltration  by 
numerous  wandering  cells.  »S  is  a  nucleus  of  the  neurilemma  sheath.  Dela- 
field's hematoxylin.     X  125 


100 


DEGENERATION  AND  REGENERATION  OF  NERVES 

ET.BEKT   CI.ABK 


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PLATE  4 

EXPLANATION  OF  FIGURES 

13  Photoinicrograijh  of  a  section  of  the  nervus  ischiadicus  of  fowl  No.  38 
— 108  days  in  regeneration  and  49  days  after  recovery  was  apparently  com- 
plete— droplets  or  globules  of  degenerated  myelin  are  to  be  seen  in  every  fiber. 
a  resembles  a  fiber  in  advanced  degeneration.  To  be  studied  in  connection  with 
figures  14,  15  antl  16,  showing  the  slowness  of  absorption  of  degenerated  myelin. 
Marchi  method.     Zeiss  2  X  16  mm. 

14  Teased  preparation  of  nervus  ischiadicus  of  fowl  No.  54 — 171  days  in 
regeneration.     Marchi  method.     Zeiss  2  X  16  mm. 

15  Teased  preparation  of  a  motor  root  of  the  nervus  ischiadicus  of  fowl 
No  57 — 1  year  and  14  days  in  regeneration — compare  with  figure  16.  Marchi 
method.     Zeiss  2  X  16  mm. 

16  Teased  preparation  of  sensory  root  of  same  nerve  as  figure  15.  Marchi 
method.     Zeiss  2  X  16  mm. 


102 


DEGENERATION  AND  REGENERATION  OF  NERVES 

ELBERT  CLARK 


PLATE  4 


13 


14 


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16 


103 


PLATE  5 

EXPLANATION  OF  FIGURES 

17  Fiber  from  the  nervus  ischiadicus  of  fowl  No.  38 — 108  days  in  regen- 
eration— a,  new  axis  cylinder;  a' ,  segment  of  old  axis  cylinder  enclosed  within 
a  large  globule  of  degenerated  myelin,  m;  n,  node  of  Ranvier.  Mallory's  phos- 
phomolybdic  acid  hematoxylin.     X  500. 

18,  19  and  20  Photomicrographs  of  sections  of  the  nervus  ischiadicus  show- 
ing new  axis  cylinders,  n,  and  fragments  of  the  old,  a',  in  globules  of  degen- 
erated myelin,  m.  Figures  18  and  19  from  fowl  No.  38.  Figure  20  from  fowl 
No.  54,  171  days  in  regeneration.  Phosphomolybdic  acid  hematoxylin.  Zeiss 
4X2  mm.  oil  immersion. 


104 


DEGENERATION  AND  REGENERATION  OF  NERVES 

ELBERT  CLARK 


PLATE  5 


19 


20 


18 


105 


PLATE  6 

EXPLANATION  OF  FIGURES 

21  Cross-section  of  fiber  of  nervus  ischiadicus  of  fowl  Xo.  o4.  A  new 
axis  cylinder,  a,  lies  adjacent  to  the  old,  a',  which  still  contains  neurofibrillae. 
m,  a  globule  of  degenerated  myelin.  Phosphomolybdic  acid  hematoxylin.  X  555. 
(See  also  fig.  23.) 

22  Cross-section  of  fiber  of  nervus  ischiadicus  of  fowl  Xo.  38.  a,  new 
axis  cylinder  with  newly  formed  medullary  sheath,  s;  a',  remnants  of  old  axis 
C3'linder;  n,  neurilemma  sheath;  c,  connective  tissue  fibrils.  Phosphomolybdic 
acid  hematoxjdin.     X  1000.     (See  also  fig.  24.) 

23,  24  and  26  Photomicrographs  respectively  of  the  same  fibers  shown  in 
figures  21,  22  and  25.     Zeiss  4X2  mm.  oil  immersion. 

25  Cross-section  of  fiber  of  nervus  ischiadicus  of  fowl  Xo.  54.  Two  new 
axis  cylinders,  a,  are  seen  within  the  old  myelin  sheath,  m,  and  in  the  position 
previously  occupied  by  the  old  axis  cylinder.  Each  has  acquired  a  secondary 
myelin  sheath.     Phosphomolybdic  acid  hematoxylin.     X  1000.     (See  also  fig.  26.) 


106 


DEGENERATION  AND  REGENERATION  OF  NERVES 

ELBERT  CLARK 


PLATE  6 


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107 


PLATE  7 

EXPLANATION   OF  FIGURES 

27  Fiber  of  the  nervus  ischiadicus  of  fowl  No.  38.  a,  new  axis  cylinder 
with  a  branch,  b,  growing  down  into  a  long  globule  of  degenerated  myelin,  vi; 
a',  remnant  of  old  axis  cylinder;  n,  nucleus  of  neurilemma  sheath;  r,  node  of 
Ranvier.  Phosphomolybdic  acid  hematoxylin.  X  500.  (See  also  figs.  28  and 
29.) 

28  and  29  Photomicrographs  at  different  focal  levels  of"  the  same  prepara- 
tion shown  in  figure  27. 

30  New  axis  cylinder,  a,  with  end  bulb,  b,  among  a  group  of  nucleated 
bands  (embryonic  nerve  fibers  or  non-meduUated  fibers?),  n,  nuclei.  Phospho- 
molybdic acid  hematoxylin.     X  555. 


108 


DEGEMERATION  AND  REGENERATION  OF  NERVES 

ELBERT   CLAEK 


PLATE    7 


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28 


27 


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109 


PLATE  8 

EXPLANATION  OF  FIGURES 

31  Photomicrograph  of  a  cross-section  of  fibers  of  the  lumbo-sacral  cord 
of  fowl  No.  38 — 108  days  in  regeneration^a,  is  a  fiber  greatly  distended  by  a 
large  globule  of  degenerated  myelin.  No  indication  of  an  axis  cylinder  is  visible. 
Phosphomolybdic  acid  hematoxylin.     Zeiss  4X2  mm.  oil  immersion. 

32  Photomicrograph  of  teased  preparation  of  nervus  ischiadicus  of  fowl 
No.  24 — 7  days  on  polished  rice.  Two  droplets  of  degenerating  myelin  are  seen 
in  fiber  a.     Marchi  method.     Zeiss  4X2  mm.  oil  immersion. 


110 


DEGENERATION  AND  REGENERATION  OF  NERVES 

ELBERT  CLARK 


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